Uses of nucleic acid encoding neuropeptide Y/peptide YY (Y2) receptors nucleic acid encoding

ABSTRACT

This invention provides isolated nucleic acid molecules encoding a human and a rat Y2 receptor, an isolated protein which is a human or rat Y2 receptor, vectors comprising an isolated nucleic acid molecule encoding a human or rat Y2 receptors, mammalian cells comprising such vectors, antibodies directed to the human or rat Y2 receptor, nucleic acid probes useful for detecting nucleic acid encoding human or rat Y2 receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human or rat Y2 receptor, pharmaceutical compounds related to human or rat Y2 receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human or rat Y2 receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human or rat Y2 receptor.

This application is a 371 of PCT/US95/01469 and a continuation-in-partof U.S. Ser. No. 08/192,288, filed Feb. 3, 1994, now U.S. Pat. No.5,545,549, the contents of which are hereby incorporated by referenceinto the subject application.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced inparenthesis by number. Full citations for these references may be foundat the end of the specification immediately preceding the claims. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the art to which thisinvention pertains.

Neuropeptides are small peptides originating from large precursorproteins synthesized by peptidergic neurons and endocrine/paracrinecells. They hold promise for treatment of neurological, psychiatric, andendocrine disorders (46). Often the precursors contain multiplebiologically active peptides. There is great diversity of neuropeptidesin the brain caused by alternative splicing of primary gene transcriptsand differential precursor processing. The neuropeptide receptors serveto discriminate between ligands and to activate the appropriate signals.

Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundantneuropeptide to be identified in mammalian brain. NPY is an importantregulator in both the central and peripheral nervous systems (47) andinfluences a diverse range of physiological parameters, includingeffects on psychomotor activity, food intake, central endocrinesecretion, and vasoactivity in the cardiovascular system. Highconcentrations of NPY are found in the sympathetic nerves supplying thecoronary, cerebral, and renal vasculature and have contributed tovasoconstriction. NPY binding sites have been identified in a variety oftissues, including spleen (48), intestinal membranes, brain (49), aorticsmooth muscle (50), kidney, testis, and placenta (2). In addition,binding sites have been reported in a number of rat and human cell lines(e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2),CHP-234, and SMS-MSN) (51,5).

Neuropeptide Y (NPY) receptor pharmacology is currently defined bystructure activity relationships within the pancreatic polypeptidefamily (1, 2). This family includes NPY, which is synthesized primarilyin neurons; peptide YY (PYY), which is synthesized primarily byendocrine cells in the gut; and pancreatic polypeptide (PP), which issynthesized primarily by endocrine cells in the pancreas. These 36 aminoacid peptides have a compact helical structure involving a "PP-fold" inthe middle of the peptide. Specific features include a polyproline helixin residues 1 through 8, a β-turn in residues 9 through 14, an α-helixin residues 15 through 30, an outward-projecting C-terminus in residues30 through 36, and a carboxyl terminal amide which appears to becritical for biological activity (3). The peptides have been used todefine at least four receptor subtypes known as Y1, Y2, Y3, and PP. Y1receptor recognition by NPY involves both N- and C-terminal regions ofthe peptide; exchange of Gln³⁴ with Pro³⁴ is fairly well tolerated (3,4, 5). Y2 receptor recognition by NPY depends primarily upon the fourC-terminal residues of the peptide (Arg³³ -Gln³⁴ -Arg³⁵ -Tyr36-NH₂)preceded by an amphipathic α-helix (3, 6, 7); exchange of Gln³⁴ withPro³⁴ is not well tolerated (4, 5). Y3 receptor recognition ischaracterized by a strong preference for NPY over PYY (8). Exchange ofGln³⁴ in NPY with Pro³⁴ is reasonably well tolerated by the Y3 receptorbut PP, which also contains Pro³⁴, does not bind well (8). The PPreceptor is reported to bind tightly to PP, less so to [Leu³¹,Pro³⁴]NPY, and even less so to NPY (3, 9). The only NPY receptor which hasbeen cloned to date is the Y1 receptor gene, from mouse (12), rat (52),and human (10). One of the key pharmacological features whichdistinguish Y1 and Y2 is the fact that the Y1 receptor (and not the Y2receptor) responds to an analog of NPY modified at residues 31 and 34([Leu31,Pro34]NPY), whereas the Y2 receptor (and not the Y1 receptor)has high affinity for the NPY peptide carboxyl-terminal fragmentNPY-(13-36) (53,4).

Receptor genes for the other two structurally related peptides, peptideYY (PYY) and pancreatic polypeptide (PP), also have not been cloned.Peptide YY occurs mainly in endocrine cells in the lowergastrointestinal tract (54). Receptors for PYY were first described inthe rat small intestine (55). This receptor has been defined asPYY-preferring because it displays a 5-10 fold higher affinity for PYYthan for NPY (55, 56). Recently, a cell line, PKSV-PCT, derived from theproximal tubules of kidneys, has been described to express receptors forPYY (57).

In the last few years only the rat and human Y1 cDNAs have been cloned(10, 11). This success was based on identifying the randomly cloned FC5"orphan receptor" (12). We now report the isolation by expressioncloning of a human hippocampal Y2 cDNA clone and two rat Y2 clones andtheir pharmacological characterization.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid molecule encoding a Y2receptor.

This invention also provides an isolated protein which is a Y2 receptor.

This invention provides a vector comprising nucleic acid encoding a Y2receptor.

This invention also provides vectors such as plasmids comprising nucleicacid encoding a Y2 receptor, adapted for expression in a bacterial cell,a yeast cell, an insect cell or a mammalian cell which additionallycomprise the regulatory elements necessary for expression of the nucleicacid in the bacterial, yeast, insect or mammalian cells operativelylinked to the nucleic acid encoding the Y2 receptor as to permitexpression thereof.

This invention provides a cell transfected with and expressing nucleicacid encoding a Y2 receptor.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of a nucleic acidmolecule encoding a Y2 receptor.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing with any sequences of an mRNAmolecule which encodes a Y2 receptor so as to prevent translation of themRNA molecule.

This invention provides an antibody directed to a Y2 receptor.

This invention provides a transgenic nonhuman mammal expressing nucleicacid encoding a Y2 receptor. This invention further provides atransgenic nonhuman mammal whose genome comprises antisense DNAcomplementary to DNA encoding a Y2 receptor so placed as to betranscribed into antisense mRNA which is complementary to mRNA encodinga Y2 receptor and which hybridizes to mRNA encoding a Y2 receptorthereby reducing its translation.

This invention further provides a transgenic nonhuman mammal comprisinga homologous recombination knockout of the native Y2 receptor.

This invention provides a method for determining whether a ligand canbind specifically to a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand under conditions permitting binding of ligands to suchreceptor, and detecting the presence of any such ligand bound to the Y2receptor, thereby determining whether the ligand binds specifically to aY2 receptor.

This invention also provides a method for determining whether a ligandis a Y2 receptor agonist which comprises contacting a cell transfectedwith and expressing nucleic acid encoding the Y2 receptor with theligand under conditions permitting the activation of a functional Y2receptor response from the cell, and detecting, by means of a bioassay,such as a second messenger assay, an increase in Y2 receptor activity,thereby determining whether the ligand is a Y2 receptor agonist.

This invention further provides a method for determining whether aligand is a Y2 receptor antagonist which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand in the presence of a known Y2 receptor agonist, such asNPY, under conditions permitting the activation of a functional Y2receptor response, and detecting, by means of a bioassay, such as asecond messenger assay, a decrease in Y2 receptor activity, therebydetermining whether the ligand is a Y2 receptor antagonist.

This invention further provides a method of screening drugs to identifydrugs which specifically bind to a Y2 receptor on the surface of a cellwhich comprises contacting a cell transfected with and expressingnucleic acid encoding the Y2 receptor with a plurality of drugs underconditions permitting binding of drugs to the Y2 receptor, anddetermining those drugs which bind to the Y2 receptor, therebyidentifying drugs which specifically bind to a Y2 receptor.

This invention also provides a method of screening drugs to identifydrugs which act as agonists of a Y2 receptor on the surface of a cellwhich comprises contacting a cell transfected with and expressingnucleic acid encoding the Y2 receptor with a plurality of drugs underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which activate the Y2 receptor,using a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor agonists.

This invention also provides a method of screening drugs to identifydrugs which act as antagonists of a Y2 receptor on the surface of a cellwhich comprises contacting a cell transfected with and expressingnucleic acid encoding the Y2 receptor with a plurality of drugs in thepresence of a known Y2 receptor agonist, such as NPY, under conditionspermitting the activation of a functional Y2 receptor response, anddetermining those drugs which inhibit the activation of the Y2 receptor,using a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor antagonists.

This invention also provides a method of detecting expression of a Y2receptor by a cell by detecting the presence of mRNA coding for the Y2receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding the Y2 receptor under hybridizingconditions, and detecting the presence of mRNA hybridized to the probe,thereby detecting the expression of a Y2 receptor by the cell.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a transgenic nonhuman mammal expressing nucleic acid encodinga Y2 receptor whose levels of Y2 receptor expression are varied by useof an inducible promoter which regulates Y2 receptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a panel of transgenic nonhuman animals each expressing nucleicacid encoding a Y2 receptor expressing nucleic acid and expressing adifferent amount of Y2 receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific Y2 receptor allelewhich comprises: a. obtaining nucleic acid of subjects suffering fromthe disorder; b. performing a restriction digest of the nucleic acidwith a panel of restriction enzymes; c. electrophoretically separatingthe resulting nucleic acid fragments on a sizing gel; d. contacting theresulting gel with a nucleic acid probe capable of specificallyhybridizing to nucleic acid encoding a Y2 receptor and labeled with adetectable marker; e. detecting labeled bands which have hybridized tothe nucleic acid encoding a Y2 receptor labelled with a detectablemarker to create a unique band pattern specific to the nucleic acid ofsubjects suffering from the disorder; f. preparing nucleic acid obtainedfor diagnosis by steps a-e; and g. comparing the unique band patternspecific to the nucleic acid of subjects suffering from the disorderfrom step e and the nucleic acid obtained for diagnosis from step f todetermine whether the patterns are the same or different and to diagnosethereby predisposition to the disorder if the patterns are the same.

This invention provides a method of preparing an isolated, purified Y2receptor which comprises constructing a vector adapted for expression ina cell which comprises the regulatory elements necessary for theexpression of nucleic acid in the cell operatively linked to the nucleicacid encoding a Y2 receptor as to permit expression thereof, wherein thecell is selected from the group consisting of bacterial cells, yeastcells, insect cells and mammalian cells; inserting the vector of theprevious step in a suitable host cell; incubating the cells underconditions allowing the expression of a Y2 receptor; recovering thereceptor so produced and purifying the receptor so recovered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Nucleotide sequence of the human hippocampal Y2 cDNA clone (SEQ.I.D. No. 1). Initiation and stop codon are indicated in bold. Onlypartial 5' and 3' untranslated sequences are shown.

FIG. 2 Deduced amino acid sequence of the human hippocampal Y2 cDNAclone encoded by the nucleotide sequence in FIG. 1 (SEQ. I.D. No. 2).

FIG. 3A through FIG. 3D

Comparison of coding nucleotide sequences between human hippocampal Y2(top row) and Y1 human cDNA clones (bottom row) (48.5% nucleotideidentity).

FIG. 4A and FIG. 4B Comparison of amino acid sequences betweenhippocampal Y2 (top row) and Y1 human cDNA clones (bottom row). (31%overall identity and 41% in the transmembrane domains).

FIG. 5A Equilibrium binding of ¹²⁵ I-PYY to membranes from COS-7 cellstransiently expressing CG-13 () and human Y1 (∘) receptors. Membraneswere incubated with ¹²⁵ I-PYY for the times indicated, in the presenceor absence of 100 nM human NPY. Specific binding, B, was plotted againsttime, t, to obtain the maximum number of equilibrium binding sites, B₁and B₂, and observed association rates, K_(obs1) and K_(obs2), accordingto the equation, B=B₁ *(1-e⁻(kobs1*t))=B₂ *(1-e⁻(kobs2*t)). Binding isshown as the percentage of total equilibrium binding, B₁ +B₂, determinedby nonlinear regression analysis. Data are representative of threeindependent experiments, with each point measured in triplicate.

FIG. 5B Equilibrium binding of ¹²⁵ I-PYY to membranes from COS-7 cellstransiently expressing CG-13 () and human Y1 (∘) receptors using thesame conditions as in FIG. 5A except for a prolonged time course of upto 180 minutes.

FIG. 6 Saturable equilibrium binding of ¹²⁵ I-PYY to membranes fromCOS-7 cells transiently expressing CG-13 receptors. Membranes wereincubated with ¹²⁵ I-PYY ranging in concentration from 0.003 nM to 2 nM,in the presence or absence of 100 nM human NPY. Specific binding, B, wasplotted against the free ¹²⁵ I-PYY concentration, [L], to obtain themaximum number of saturable binding sites, B_(max), and the ¹²⁵ I-PYYequilibrium dissociation constant, K_(d), according to the bindingisotherm, B=B_(max) [L]/([L]+K_(d)). Specific binding is shown. Data arerepresentative of three independent experiments, with each pointmeasured in triplicate.

FIG. 7A Competitive displacement of ¹²⁵ I-PYY on membranes from COS-7cells transiently expressing Human Y1 receptors. Membranes wereincubated with ¹²⁵ I-PYY and increasing concentrations of peptidecompetitors. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the equation, K_(i) =IC₅₀ /(1+[L]/K_(d)), where [L]is the ¹²⁵ I-PYY concentration and K_(d) is the equilibrium dissociationconstant of ₁₂₅ I-PYY. Data are representative of at least twoindependent experiments, with each point measured once or in duplicate.Rank orders of affinity for these and other compounds are listedseparately in Table 2.

FIG. 7B Competitive displacement of ¹²⁵ I-PYY on membranes from COS-7cells transiently expressing human Y2 receptors. Membranes wereincubated with ¹²⁵ I-PYY and increasing concentrations of peptidecompetitors. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the equation, K_(i) =IC₅₀ /(1+[L]/K_(d)), where [L]is the ¹²⁵ I-PYY concentration and K_(d) is the equilibrium dissociationconstant of 125-PYY. Data are representative of at least two independentexperiments, with each point measured once or in duplicate. Rank ordersof affinity for these and other compounds are listed separately in Table2.

FIGS. 8A-8E Nucleotide sequence (SEQ. I.D. No. 3) and deduced amino acidsequence (SEQ. I.D. No. 4) of the rat Y2 receptor encoded by rs5a.Nucleotides are presented in the 5' to 3' orientation and the codingregion is numbered starting from the putative initiating methionine andending in the termination codon. Deduced amino acid sequence bytranslation of a long open reading frame is shown using one-lettersymbols.

FIGS. 9A-9D Nucleotide sequence (SEQ. I.D. No. 5) and deduced amino acidsequence (SEQ. I.D. No. 6) of the rat Y2 receptor encoded by rs26a.Nucleotides are presented in the 5' to 3' orientation and the codingregion is numbered starting from the putative initiating methionine andending in the termination codon. Deduced amino acid sequence bytranslation of a long open reading frame is shown using one-lettersymbols.

FIGS. 10A-10D Alignment of rat and human Y2 receptors: nucleotidesequences. Nucleotide sequences of the coding regions of the human Y2receptor (HumY2) and the rat Y2 receptors encoded by rs5a (RatY2a) andrs26a (RatY2b) are shown; the nucleotide sequence of rs26a (RatY2b) isidentical to rs5a (RatY2a) except where shown. Rat and human Y2nucleotide sequences exhibit ˜86% identity in the coding region.

FIGS. 11A-11B Alignment of rat and human Y2 receptors: amino acidsequences. Complete predicted amino acid sequences of the human Y2receptor (Hum Y2) and the rat Y2 receptor encoded by rs5a (Rat Y2a) areshown; the amino acid sequence of RatY2b encoded by rs26a is identicalto RatY2a except where shown. Rat and human Y2 amino acid sequences are˜94% identical overall and ˜98% identical in the transmembrane domains(bracketed). Single letter abbreviations for amino acids are shown.

FIGS. 12A-12H Localization of Rat Y2 mRNA in the rat central nervoussystem. Schematic diagrams of half-coronal sections through the ratbrain showing the distribution of neuropeptide Y Y2 receptor mRNAobtained with radiolabelled oligonucleotide probes and in situhybridization histochemistry. The stars show the location of labeledneuronal populations, and are not indicative of the number of cellsobserved in each area.

FIGS. 13A-13B Effects of Gpp(NH)p on radio ligand binding to Y2receptors. Binding data were generated from competitive displacementassays in the absence () or presence (∘) of 100 μM Gpp(NH)p. Themaximum specific binding detected under control conditions (in theabsence of Gpp(NH)p) was used to normalize the data. A) Human Y2receptor transiently expressed in COS-7 cells. B) Rat Y2a receptortransiently expressed in COS-7 cells.

FIG. 14 Inhibition of forskolin-stimulated cAMP accumulation in intactcells stably expressing the human Y2 receptor. Functional data werederived from radioimmunoassay of cAMP in 293 cells stimulated with 10 μMforskolin over a 5 min period. Human PYY was tested for agonist activityover the same period. Data were fit to a four parameter logisticequation by nonlinear regression. Data generated from stably transfected293 cells () and from stably transfected NIH-3T3 cells (∘). Data shownare representative of ten () and two (∘) independent experiments.

FIGS. 15A-15C Stimulation of intracellular free calcium concentration inintact 293 cells stably expressing the human Y2 receptor. A) Timecourse. Functional data were derived from Fura-2/AM fluorescence in 293cells stimulated with 1 μM human PYY at the time indicated by the arrow.B) Time course. Cells were stimulated with 1 μM human PYY as in A exceptthat 1 mM EGTA was included in the extracellular solution. C)Concentration/response curve for PYY-dependent mobilization ofintracellular calcium in 293 cells stably transfected with the human Y2receptor. Data were fit to a four parameter logistic equation bynonlinear regression. Data shown are representative of at least twoindependent experiments.

FIG. 16. Northern analysis of various human brain areas. Hybridizationwas done under conditions of high stringency, as described inExperimental Details. The probe was a ³² P-labeled DNA fragment(specific activity 3×10⁹ cpm/μg) corresponding to the entire codingregion (as shown in FIG. 10) of the human NPY Y2 receptor. The BRL RNAladder was used as molecular weight markers.

FIG. 17. Southern analysis of genomic DNA encoding the human NPY Y2receptor subtype. Hybridization was done under conditions of highstringency, as described in Experimental Details. The probe was a ³²P-labeled DNA fragment (specific activity 2.5×10⁹ cpm/μg) correspondingto the TM1-TM5 region of the human NPY Y2 receptor (as shown in FIG.11). Hind III digested λ DNA was used as molecular weight markers.

FIGS. 18A-18D Photomicrographs showing some of the controls used for NPYY2 oligonucleotide probe specificity (A, B), and tissue distribution ofthe hybridization signal in rat brain (C, D). A. Darkfieldphotomicrograph of the hybridization signal obtained using theradiolabeled antisense probe on COS-7 cells transfected with the rat Y2DNA. B. Hybridization signal obtained following hybridization with theradiolabeled sense probe, also on transfected COS-7 cells. Only theantisense probes hybridize to the transfected cells. C. Brightfieldphotomicrograph of the hybridization signal observed in the CA3 regionof the rat hippocampus. Silver grains are found over neuronal cellbodies (arrows) in the pyramidal cell layer (sp), but not over thestratum lucidum (slu) or stratum radiatum (sr). D. Hybridization signalobserved over neurons (arrows) in the arcuate nucleus of thehypothalamus. The darkly stained ependymal lining of the third ventriclecan be seen to the left of the micrograph (asterisk).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

C=cytosine

A=adenine

T=thymine

G=guanine

This invention provides isolated nucleic acid molecules which encode Y2receptors. In one embodiment, the Y2 receptor encoded is a human Y2receptor. In another embodiment, the Y2 receptor encoded is a rat Y2receptor. As used herein, the term Y2 receptor encompasses any aminoacid sequence, polypeptide or protein having substantially the samepharmacology provided for the subject human Y2 receptor as shown inTables 2-4 and FIGS. 5A-7B. As described herein our cloned receptor hasa Y2 pharmacological profile that differs from the NPY receptor subtypesY1 and Y3, PYY receptor, and PP receptor, and is therefore designated asthe Y2 receptor. The only NPY receptor which has been cloned to date isthe Y1 receptor gene, from mouse (Eva et al., 1992), rat (Eva et al.,1990), and human (Larhammar et al., 1992). The human Y2 receptor'sgreatest homology with any known receptor disclosed in the Genbank/EMBLdatabases is a 42% overall amino acid identity with the human Y1receptor.

This invention provides isolated nucleic acid molecules encoding Y2receptors. In one embodiment, the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor. As usedherein, the term "isolated nucleic acid molecule" means a nucleic acidmolecule that is a molecule in a form which does not occur in nature.Examples of such an isolated nucleic acid molecule are an RNA, cDNA, orisolated genomic DNA molecule encoding a Y2 receptor. The human Y2receptor has an amino acid sequence substantially the same as thededuced amino acid sequence shown in FIG. 2 and any human receptorhaving substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 is by definition a human Y2 receptor. The ratY2 receptor has an amino acid sequence substantially the same as thededuced amino acid sequences shown in FIG. 8 or FIG. 9. One means ofisolating another Y2 receptor is to probe a genomic library with anatural or artificially designed DNA probe, using methods well known inthe art. DNA probes derived from the human and the rat receptor Y2 geneare particularly useful probes for this purpose. DNA and cDNA moleculeswhich encode Y2 receptors may be used to obtain genomic DNA, cDNA or RNAfrom human, mammalian or other animal sources, or to isolate relatedcDNA or genomic clones by the screening of cDNA or genomic libraries bymethods described in more detail below. Transcriptional regulatoryelements from the 5' untranslated region of the isolated clones, andother stability, processing, transcription, translation, and tissuespecificity-determining regions from the 3' and 5' untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding aY2 receptor. Such molecules may have coding sequences substantially thesame as the coding sequences shown in FIGS. 1, 8 and 9. The DNA moleculeof FIG. 1 encodes the sequence of the human Y2 receptor gene. The DNAmolecules of FIGS. 8 and 9 encode the sequence of two rat Y2 receptorgenes.

This invention further provides DNA molecules encoding Y2 receptorshaving coding sequences substantially the same as the coding sequencesshown in FIGS. 1, 8 and 9. These molecules are obtained by the meansdescribed above.

This invention also provides an isolated nucleic acid molecule encodinga Y2 receptor wherein the nucleic acid molecule encodes a receptor beingcharacterized by an amino acid sequence in the transmembrane region,which amino acid sequence has 60% homology or higher to the amino acidsequence in the transmembrane region of the human Y2 receptor as shownin FIG. 11.

This invention also provides purified isolated proteins which are Y2receptors. In one embodiment, the Y2 receptor protein is a human Y2receptor protein. In another embodiment, the Y2 receptor protein is arat Y2 receptor protein. As used herein, the term "isolated protein"means a protein molecule free of other cellular components. Examples ofsuch proteins are isolated proteins having substantially the same aminoacid sequence as the amino acid sequences shown in FIGS. 2, 8, and 9,which are a human Y2 receptor and two rat Y2 receptors, respectively.One means for obtaining an isolated Y2 receptor is to express DNAencoding the receptor in a suitable host, such as a bacterial, yeast,insect or mammalian cell, using methods well known in the art, andrecovering the receptor protein after it has been expressed in such ahost, again using methods well known in the art. The receptor may alsobe isolated from cells which express it, in particular from cells whichhave been transfected with the expression vectors described below inmore detail.

This invention provides vectors comprising nucleic acid molecules suchas DNA, RNA, or cDNA encoding Y2 receptors. In one embodiment, thenucleic acid encodes a human Y2 receptor. In another embodiment, thenucleic acid encodes a rat Y2 receptor. Examples of vectors are virusessuch as bacteriophages (such as phage lambda), animal viruses (such asHerpes virus, Murine Leukemia virus, and Baculovirus), cosmids, plasmids(such as pUC18, available from Pharmacia, Piscataway, N.J.), and otherrecombination vectors. Nucleic acid molecules are inserted into vectorgenomes by methods well known in the art. For example, insert and vectorDNA can both be exposed to a restriction enzyme to create complementaryends on both molecules which base pair with each other and are thenligated together with a ligase. Alternatively, linkers can be ligated tothe insert DNA which correspond to a restriction site in the vector DNA,which is then digested with the restriction enzyme which cuts at thatsite. Other means are also available. Specific examples of such plasmidsare: a plasmid comprising cDNA having a coding sequence substantiallythe same as the coding sequence shown in FIG. 1 and designated cloneCG-13 (Seq. I.D. No. 1); or a plasmid comprising genomic DNA having acoding sequence substantially the same as the coding sequence shown inFIG. 8 and designated clone rS5a (Seq. I.D. No. 3), or the codingsequence shown in FIG. 9 and designated clone rS26a (Seq. I.D. No. 5).

This invention also provides vectors comprising nucleic acid moleculesencoding Y2 receptors, adapted for expression in a bacterial cell, ayeast cell, an insect cell or a mammalian cell which additionallycomprise the regulatory elements necessary for expression of the nucleicacid in the bacterial, yeast, insect or mammalian cells operativelylinked to the nucleic acid encoding a Y2 receptor as to permitexpression thereof. Nucleic acid having coding sequences substantiallythe same as the coding sequence shown in FIG. 1 may be usefully insertedinto the vectors to express human Y2 receptors. Nucleic acid havingcoding sequences substantially the same as the coding sequences shown inFIGS. 8 and 9 may be usefully inserted into vectors to express rat Y2receptors. Regulatory elements required for expression include promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes a promoter such as the lac promoter and for transcriptioninitiation the Shine-Dalgarno sequence and the start codon AUG(Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory,1982). Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Furthermore, an insect expression vector,such as recombinant baculovirus, uses the polyhedron gene expressionsignals for expression of the inserted gene in insect cells. Suchvectors may be obtained commercially or assembled from the sequencesdescribed by methods well known in the art, for example the methodsdescribed above for constructing vectors in general. Expression vectorsare useful to produce cells that express the receptor. Certain uses forsuch cells are described in more detail below.

This invention further provides a plasmid adapted for expression in abacterial cell, a yeast cell, an insect cell, or, in particular, amammalian cell which comprises a nucleic acid molecule encoding a Y2receptor and the regulatory elements necessary for expression of thenucleic acid in the bacterial, yeast, insect, or mammalian celloperatively linked to the nucleic acid encoding the Y2 receptor as topermit expression thereof. In one embodiment, the Y2 receptor is a humanY2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. Some plasmids adapted for expression in a mammalian cell arepSVL (available from Pharmacia, Piscataway, N.J.) and pcEXV-3 (73). Onespecific example of such a plasmid is a plasmid adapted for expressionin a mammalian cell comprising cDNA having a coding sequencesubstantially the same as the coding sequence shown in FIG. 1 and theregulatory elements necessary for expression of the DNA in the mammaliancell which is designated pcEXV-hY2, deposited on Jan. 27, 1994 underATCC Accession No. 75659. Other specific examples of such plasmids areplasmids adapted for expression in a mammalian cell comprising genomicDNA having coding sequences substantially the same as the codingsequences shown in FIGS. 8 and 9 and the regulatory elements necessaryfor expression of the DNA in the mammalian cell which are designatedpcEXV-rY2a, deposited on Jan. 25, 1995 under ATCC Accession No. 97035;and pcEXV-rY2b, deposited on Jan. 25, 1995 under ATCC Accession No.97036, respectively. Those skilled in the art will readily appreciatethat numerous plasmids adapted for expression in a mammalian cell whichcomprise DNA encoding Y2 receptors and the regulatory elements necessaryto express such DNA in the mammalian cell may be constructed utilizingexisting plasmids and adapted as appropriate to contain the regulatoryelements necessary to express the DNA in the mammalian cell. Theplasmids may be constructed by the methods described above forexpression vectors and vectors in general, and by other methods wellknown in the art.

The deposits discussed supra, and the other deposits discussed herein,were made pursuant to, and in satisfaction of, the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

This invention provides a cell transfected with and expressing nucleicacid encoding a Y2 receptor. In one embodiment the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. An example of such a cell is a mammalian cell transfected witha plasmid adapted for expression in a mammalian cell, which comprisesnucleic acid encoding a Y2 receptor, and the regulatory elementsnecessary for expression of the nucleic acid in the mammalian celloperatively linked to the nucleic acid encoding a Y2 receptor as topermit expression thereof; the protein encoded thereby expressed on thecell surface. Numerous mammalian cells may be used as hosts, including,for example, the mouse fibroblast cell NIH-3T3, CHO cells, HeLa cells,LM(tk-) cells, etc. Expression plasmids such as that described supra maybe used to transfect cells by methods well known in the art such ascalcium phosphate precipitation, or DNA encoding these Y2 receptors maybe otherwise introduced into cells, e.g., by microinjection, to obtainmammalian cells which comprise nucleic acid, e.g., cDNA or a plasmid,encoding a Y2 receptor. A specific example of such cells is a cellcomprising the pcEXV-hY2 plasmid adapted for expression in a mammaliancell comprising cDNA encoding the Y2 receptor and the regulatoryelements necessary for expression of the DNA in the mammalian cell,which is designated 293-hY2-10 and deposited on Jan. 27, 1994 under ATCCAccession No. 11837. Another specific example of such cells is a cellcomprising the pcEXV-hY2 plasmid adapted for expression in a mammaliancell comprising cDNA encoding the Y2 receptor and the regulatoryelements necessary for expression of the DNA in the mammalian cell,which is designated N-hY2-5 and deposited on Jan. 25, 1995 under ATCCAccession No. CRL-11825.

This invention provides a method for determining whether a ligand canbind specifically to a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding a Y2 receptor, theprotein encoded thereby is expressed on the cell surface, with theligand under conditions permitting binding of ligands known to bind tothe Y2 receptor, and detecting the presence of any of the ligand boundto the Y2 receptor, thereby determining whether the ligand bindsspecifically to the Y2 receptor. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

This invention further provides a method for determining whether aligand can bind specifically to a Y2 receptor, which comprisescontacting a cell transfected with and expressing nucleic acid encodingthe Y2 receptor with the ligand under conditions permitting binding ofligands to such receptor, and detecting the presence of any such ligandbound to the Y2 receptor, wherein the Y2 receptor is characterized by anamino acid sequence in the transmembrane region, such amino acidsequence having 60% homology or higher to the amino acid sequence in thetransmembrane region of the Y2 receptor shown in FIG. 11. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

This invention provides a method for determining whether a ligand canbind specifically to a Y2 receptor which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the ligand with the membrane fraction from the cell extractunder conditions permitting binding of ligands to such receptor, anddetecting the presence of any ligand bound to the Y2 receptor, therebydetermining whether the compound is capable of binding specifically to aY2 receptor. In one embodiment, the Y2 receptor is a human Y2 receptor.In another embodiment, the Y2 receptor is a rat Y2 receptor.

This invention also provides a method for determining whether a ligandis a Y2 receptor agonist. As used herein, the term "agonist" means anyligand capable of increasing Y2 receptor functional activity. Thiscomprises contacting a cell transfected with and expressing nucleic acidencoding a Y2 receptor with the ligand under conditions permitting theactivation of a functional Y2 receptor response from the cell, anddetecting, by means of a bioassay, such as a second messenger assay, anincrease in Y2 receptor activity, thereby determining whether the ligandacts as a Y2 receptor agonist. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

This invention further provides a method for determining whether aligand is a Y2 receptor agonist which comprises preparing a cell extractfrom cells transfected with and expressing nucleic acid encoding a Y2receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction of the extract with the ligand underconditions permitting the activation of a functional Y2 receptorresponse, and detecting, by means of a bioassay, such as a secondmessenger assay, an increase in Y2 receptor activity, therebydetermining whether the ligand is a Y2 receptor agonist. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

This invention also provides a method for determining whether a ligand aY2 receptor antagonist. As used herein, the term "antagonist" means anyligand capable of decreasing Y2 receptor functional activity. Thiscomprises contacting a cell transfected with and expressing nucleic acidencoding a Y2 receptor with the ligand in the presence of a known Y2receptor agonist such as NPY, under conditions permitting the activationof a functional Y2 receptor response, and detecting, by means of abioassay, such as a second messenger assay, a decrease in Y2 receptoractivity, thereby determining whether the ligand is a Y2 receptorantagonist. In one embodiment, the Y2 receptor is a human Y2 receptor.In another embodiment, the Y2 receptor is a rat Y2 receptor.

This invention also provides a method for determining whether a ligandis a Y2 receptor antagonist which comprises preparing a cell extractfrom cells transfected with and expressing nucleic acid encoding a Y2receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction of the extract with the ligand in thepresence of a known Y2 receptor agonist, such as NPY, under conditionspermitting the activation of a functional Y2 receptor response, anddetecting, by means of a bioassay, such as a second messenger assay, adecrease in Y2 receptor activity, thereby determining whether the ligandis a Y2 receptor antagonist. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

In one embodiment, the second messenger assays referred to comprisemeasurement of intracellular cAMP. In another embodiment, the secondmessenger assays comprise measurement of intracellular calciummobilization.

In one embodiment, the nucleic acid in the cells referred to aboveencodes a Y2 receptor having an amino acid sequence substantially thesame as the amino acid sequence shown in FIG. 2. In another embodiment,the nucleic acid in the cells referred to above encodes a Y2 receptorhaving an amino acid sequence substantially the same as the amino acidsequences shown in FIG. 8 or FIG. 9. In one embodiment, the cell is amammalian cell. Preferably, the mammalian cell is non-neuronal inorigin. An example of a nonneuronal mammalian cell is a COS-7 cell.Other examples of a non-neuronal mammalian cells that can be used forfunctional assays with Y2 receptors are the 293 human embryonic kidneycells, mouse embryonic fibroblast NIH-3T3 cells, and LM(tk-) cells.

The preferred method for determining whether a ligand is capable ofbinding specifically to a Y2 receptor comprises contacting a transfectednonneuronal mammalian cell (i.e. a cell that does not naturally expressany type of NPY, PP, or PYY receptor, and thus will only express such areceptor if it is transfected into the cell) expressing a Y2 receptor onits surface, or contacting a membrane preparation derived from such atransfected cell, with the ligand under conditions which are known toprevail, and thus to be associated with, in vivo binding of the ligandto and/or activation of a Y2 receptor, and detecting the presence of anyof the ligand being tested bound to the Y2 receptor on the surface ofthe cell, or detecting activation of the Y2 receptor, therebydetermining whether the ligand binds to, activates or inhibits theactivation of the Y2 receptor. Activation of a Y2 receptor may bedetected by means of a second messenger assay. Such a response system isobtained by transfection of nucleic acid into a suitable host cellcontaining the desired second messenger system such as phospholipase C,adenylate cyclase, guanylate cyclase or ion channels. A suitable hostcell can be isolated from pre-existing cell lines, or can be generatedby inserting appropriate components of second messenger systems intoexisting cell lines. Such a transfected cell provides a completeresponse system for investigation or assay of the activity of Y2receptors with ligands as described above. Transfection systems areuseful as living cell cultures for competitive binding assays betweenknown or candidate drugs and ligands which bind to the receptor andwhich are labeled by radioactive, spectroscopic or other reagents.Membrane preparations containing the receptor isolated from transfectedcells are also useful for Y2 receptor activity and competitive bindingassays. Functional assays of signal transduction pathways intransfection systems determine a ligand's efficacy of activating thereceptor. A transfection system constitutes a "drug discovery system"useful for the identification of natural or synthetic compounds withpotential for drug development that can be further modified or useddirectly as therapeutic compounds to activate or inhibit the naturalfunctions of the Y2 receptor. The transfection system is also useful fordetermining the affinity and efficacy of known drugs at the Y2 receptorsites.

This invention provides a pharmaceutical composition comprising aneffective amount of the Y2 receptor agonist determined by the methodsdescribed above and a pharmaceutically acceptable carrier. As usedherein, the term "pharmaceutically acceptable carrier" encompasses anyof the standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. In a further embodiment, the Y2 receptor agonistis not previously known.

This invention further provides a pharmaceutical composition comprisingan effective amount of the Y2 receptor antagonist determined by themethods described above and a pharmaceutically acceptable carrier. Inone embodiment the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. In a furtherembodiment, the Y2 receptor antagonist is not previously known.

This invention also provides a method of screening drugs to identifydrugs which specifically bind to a Y2 receptor on the surface of a cellwhich comprises contacting a cell transfected with and expressingnucleic acid encoding the Y2 receptor with a plurality of drugs underconditions permitting binding of drugs to the Y2 receptor, anddetermining those drugs which bind specifically to the cell, therebyidentifying drugs which specifically bind to a Y2 receptor. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

This invention also provides a method of screening drugs to identifydrugs which specifically bind to a Y2 receptor on the surface of a cellwhich comprises preparing a cell extract from the cells transfected withand expressing nucleic acid encoding the Y2 receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with a plurality of drugs under conditions permitting bindingof drugs to the Y2 receptor, and determining those drugs which bindspecifically to the transfected cell, thereby identifying drugs whichbind specifically to a Y2 receptor. In one embodiment, the Y2 receptoris a human Y2 receptor. In another embodiment, the Y2 receptor is a ratY2 receptor.

This invention also provides a method of screening drugs to identifydrugs which act as Y2 receptor agonists which comprises contacting acell transfected with and expressing nucleic acid encoding a Y2 receptorwith a plurality of drugs under conditions permitting the activation ofa functional Y2 receptor response, and determining those drugs whichactivate such Y2 receptor, using a bioassay, such as a second messengerassay, thereby identifying drugs which act as Y2 receptor agonists. Inone embodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment the Y2 receptor is a rat Y2 receptor. In a furtherembodiment, the Y2 receptor agonist is not previously known.

This invention provides a method of screening drugs to identify drugswhich act as agonists of a Y2 receptor which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with a plurality of drugs underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which activate such receptor,using a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor agonists. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. In a further embodiment, the Y2 receptor agonistis not previously known.

This invention also provides a method of screening drugs to identifydrugs which as Y2 receptor antagonists which comprises contacting a celltransfected with and expressing nucleic acid encoding a Y2 receptor witha plurality of drugs in the presence of a known Y2 receptor agonist suchas NPY under conditions permitting the activation of a functional Y2receptor response, and determining those drugs which inhibit theactivation of the receptor, using a bioassay, such as a second messengerassay, thereby identifying drugs which act as Y2 receptor antagonists.In one embodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. In a furtherembodiment, the Y2 receptor antagonist is not previously known.

This invention provides a method of screening drugs to identify drugswhich act as Y2 receptor antagonists which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with a plurality of drugs in thepresence of a known Y2 receptor agonist, such as NPY, under conditionspermitting the activation of a functional Y2 receptor response, anddetermining those drugs which inhibit the activation of the receptorusing a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor antagonists. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. In a further embodiment, the Y2 receptorantagonist is not previously known.

In one embodiment of the above described methods, the second messengerassay comprises measurement of intracellular cAMP. In anotherembodiment, the second messenger assay comprises measurement ofintracellular calcium mobilization.

The nucleic acid in the cells of the methods described above may have acoding sequence substantially the same as the coding sequences shown inFIGS. 1, 8 and 9. Preferably, the mammalian cell is nonneuronal inorigin. An example of a nonneuronal mammalian cell is an COS-7 cell.Other examples of a non-neuronal mammalian cell to be used forfunctional assays are 293 human embryonic kidney cells, mouse embryonicfibroblast NIH-3T3 cells and LM(tk-) cells. Drug candidates areidentified by choosing chemical compounds which bind with high affinityto the expressed Y2 receptor protein in transfected cells, usingradioligand binding methods well known in the art, examples of which areshown in the binding assays described herein. Drug candidates are alsoscreened for selectivity by identifying compounds which bind with highaffinity to the Y2 receptor but do not bind with high affinity to anyother NPY receptor subtype or to any other known receptor site. Becauseselective, high affinity compounds interact primarily with the target Y2receptor site after administration to the patient, the chances ofproducing a drug with unwanted side effects are minimized by thisapproach.

This invention provides a pharmaceutical composition comprising aneffective amount of a drug identified by the methods described above anda pharmaceutically acceptable carrier.

As used herein, an "effective amount" is an amount of the drug effectiveto produce the desired result in a subject when administered inaccordance with the chosen regimen. Once the candidate drug has beenshown to be adequately bio-available following a particular route ofadministration, for example orally or by injection (adequate therapeuticconcentrations must be maintained at the site of action for an adequateperiod to gain the desired therapeutic benefit), and has been shown tobe non-toxic and therapeutically effective in appropriate diseasemodels, the drug may be administered to patients by that route ofadministration determined to make the drug bio-available, in anappropriate solid or solution formulation, to gain the desiredtherapeutic benefit.

This invention also provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by activation of a Y2receptor which comprises administering to a subject an effective amountof the pharmaceutical composition described above, thereby treating theabnormality. In one embodiment, the Y2 receptor is a human Y2 receptor.In another embodiment, the Y2 receptor is a rat Y2 receptor.

As used herein, the term "effective amount" means that amount of a drugwhich is able to produce the desired result in a subject whenadministered in accordance with the chosen regimen. Typically, aneffective amount is an amount from about 0.01 mg per subject per day toabout 500 mg per subject per day. More typically this amount is anamount from about 0.1 mg per subject per day to about 60 mg per subjectper day. Most typically, this amount is an amount from about 1 mg persubject per day to about 20 mg per subject per day. Optimal dosages tobe administered may be determined by those skilled in the art, and willvary with the particular drug in use, the strength of the preparation,the mode of administration, and the advancement of the diseasecondition. Additional factors depending on the particular subject beingtreated will result in a need to adjust dosages, including subject age,weight, gender, diet, and time of administration.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by activation of a Y2 receptorwhich comprises administering to a subject an effective amount of a Y2receptor agonist determined by the methods described above, therebytreating the abnormality. In one embodiment, the Y2 receptor is a humanY2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

This invention further provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to a subject aneffective amount of the pharmaceutical composition described above,thereby treating the abnormality. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

This invention also provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan effective amount of a Y2 receptor antagonist determined by themethods described above, thereby treating the abnormality. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of a nucleic acidmolecule encoding a Y2 receptor, for example with a coding sequenceincluded within the sequences shown in FIGS. 1, 8 and 9. As used herein,the phrase "specifically hybridizing" means the ability of a nucleicacid molecule to recognize a nucleic acid sequence complementary to itsown and to form double-helical segments through hydrogen bonding betweencomplementary base pairs. As used herein, a "unique sequence" is asequence specific to only the nucleic acid molecules encoding the Y2receptor. In one embodiment the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor. Nucleic acidprobe technology is well known to those skilled in the art who willreadily appreciate that such probes may vary greatly in length and maybe labeled with a detectable label, such as a radioisotope orfluorescent dye, to facilitate detection of the probe. Detection ofnucleic acid encoding Y2 receptors is useful as a diagnostic test forany disease process in which levels of expression of the correspondingY2 receptor is altered. DNA probe molecules are produced by insertion ofa DNA molecule which encodes Y2 receptor or fragments thereof intosuitable vectors, such as plasmids or bacteriophages, followed byinsertion into suitable bacterial host cells and replication andharvesting of the DNA probes, all using methods well known in the art.For example, the DNA may be extracted from a cell lysate using phenoland ethanol, digested with restriction enzymes corresponding to theinsertion sites of the DNA into the vector (discussed above),electrophoresed, and cut out of the resulting gel. Examples of such DNAmolecules are shown in FIGS. 1, 8 and 9. The probes are useful for `insitu` hybridization or in order to locate tissues which express thisgene family, or for other hybridization assays for the presence of thesegenes or their mRNA in various biological tissues. In addition,synthesized oligonucleotides (produced by a DNA synthesizer)complementary to the sequence of a DNA molecule which encodes a Y2receptor are useful as probes for these genes, for their associatedmRNA, or for the isolation of related genes by homology screening ofgenomic or cDNA libraries, or by the use of amplification techniquessuch as the Polymerase Chain Reaction. Synthesized oligonucleotides asdescribed may also be used to determine the cellular localization of themRNA produced by the Y2 gene by in situ hybridization.

This invention also provides a method of detecting expression of a Y2receptor by detecting the presence of mRNA coding for a Y2 receptorwhich comprises obtaining total mRNA from the cell using methods wellknown in the art and contacting the mRNA so obtained with a nucleic acidprobe comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of a nucleic acid molecule encoding the Y2 receptor underhybridizing conditions, and detecting the presence of mRNA hybridized tothe probe, thereby detecting the expression of the Y2 receptor by thecell. In one embodiment, the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor. Hybridizationof probes to target nucleic acid molecules such as mRNA moleculesemploys techniques well known in the art. In one possible means ofperforming this method, nucleic acids are extracted by precipitationfrom lysed cells and the mRNA is isolated from the extract using acolumn which binds the poly-A tails of the mRNA molecules. The mRNA isthen exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to an mRNA molecule which encodes aY2 receptor so as to prevent translation of the mRNA molecule. Theantisense oligonucleotide may have a sequence capable of specificallyhybridizing with the cDNA molecule whose sequence is shown in FIG. 1, orwith the genomic DNA molecule whose sequences are shown in FIGS. 8 and9. A particular example of an antisense oligonucleotide is an antisenseoligonucleotide comprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising anamount of the oligonucleotide described above effective to decreaseactivity of a Y2 receptor by passing through a cell membrane andspecifically hybridizing with mRNA encoding a Y2 receptor in the cell soas to prevent its translation and a pharmaceutically acceptable carriercapable of passing through a cell membrane. The oligonucleotide may becoupled to a substance which inactivates mRNA, such as a ribozyme. Thepharmaceutically acceptable carrier capable of passing through cellmembranes may also comprise a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type. The structure may be part of a protein known tobind a cell-type specific receptor, for example an insulin molecule,which would target pancreatic cells. In one embodiment, the Y2 receptoris a human Y2 receptor. In another embodiment, the Y2 receptor is a ratY2 receptor. DNA molecules having coding sequences substantially thesame as the coding sequences shown in FIGS. 1, 8 and 9 may be used asthe oligonucleotides of the pharmaceutical composition.

This invention also provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a Y2 receptor which comprises administering to the subject aneffective amount of the pharmaceutical composition described above,thereby treating the abnormality. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. Several examples of such abnormalities are hypertension,gastrointestinal disorders, epilepsy, sleep disorders, and cognitivedisorders, (58-80).

Antisense oligonucleotide drugs inhibit translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures are designed to bind to mRNA encoding the Y2 receptor andinhibit translation of mRNA and are useful as drugs to inhibitexpression of Y2 receptor genes in patients. This invention provides ameans to therapeutically alter levels of expression of Y2 receptors bythe use of a synthetic antisense oligonucleotide drug (SAOD) whichinhibits translation of mRNA encoding these receptors. Syntheticoligonucleotides, or other antisense chemical structures designed torecognize and selectively bind to mRNA, are constructed to becomplementary to portions of the nucleotide sequences shown in FIGS. 1,8, and 9 of DNA, RNA or of chemically modified, artificial nucleicacids. The SAOD is designed to be stable in the blood stream foradministration to patients by injection, or in laboratory cell cultureconditions, for administration to cells removed from the patient. TheSAOD is designed to be capable of passing through cell membranes inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SAOD which render it capable of passingthrough cell membranes (e.g. by designing small, hydrophobic SAODchemical structures) or by virtue of specific transport systems in thecell which recognize and transport the SAOD into the cell. In addition,the SAOD can be designed for administration only to certain selectedcell populations by targeting the SAOD to be recognized by specificcellular uptake mechanisms which binds and takes up the SAOD only withincertain selected cell populations. For example, the SAOD may be designedto bind to a receptor found only in a certain cell type, as discussedabove. The SAOD is also designed to recognize and selectively bind tothe target mRNA sequence, which may correspond to a sequence containedwithin the sequences shown in FIGS. 1, 8, and 9 by virtue ofcomplementary base pairing to the mRNA. Finally, the SAOD is designed toinactivate the target mRNA sequence by any of three mechanisms: 1) bybinding to the target mRNA and thus inducing degradation of the mRNA byintrinsic cellular mechanisms such as RNAse I digestion, 2) byinhibiting translation of the mRNA target by interfering with thebinding of translation-regulating factors or of ribosomes, or 3) byinclusion of other chemical structures, such as ribozyme sequences orreactive chemical groups, which either degrade or chemically modify thetarget mRNA. Synthetic antisense oligonucleotide drugs have been shownto be capable of the properties described above when directed againstmRNA targets (74,75). In addition, coupling of ribozymes to antisenseoligonucleotides is a promising strategy for inactivating target mRNA(76). An SAOD serves as an effective therapeutic agent if it is designedto be administered to a patient by injection, or if the patient's targetcells are removed, treated with the SAOD in the laboratory, and replacedin the patient. In this manner, an SAOD serves as a therapy to reducereceptor expression in particular target cells of a patient, in anyclinical condition which may benefit from reduced expression of Y2receptors.

This invention provides an antibody directed to a Y2 receptor, forexample, a monoclonal antibody directed to an epitope of a Y2 receptorpresent on the surface of a cell and having an amino acid sequencesubstantially the same as an amino acid sequence for a cell surfaceepitope of the Y2 receptor included in the amino acid sequences shown inFIGS. 2, 8 and 9 (Seq. I.D. Nos. 2, 4, and 6, respectively). In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. Amino acid sequencesmay be analyzed by methods well known in the art to determine whetherthey produce hydrophobic or hydrophilic regions in the proteins whichthey build. In the case of cell membrane proteins, hydrophobic regionsare well known to form the part of the protein that is inserted into thelipid bilayer which forms the cell membrane, while hydrophilic regionsare located on the cell surface, in an aqueous environment. Thereforeantibodies to the hydrophilic amino acid sequences shown in FIGS. 2, 8,and 9 will probably bind to a surface epitope of a Y2 receptor, asdescribed. Antibodies directed to Y2 receptors may be serum-derived ormonoclonal and are prepared using methods well known in the art. Forexample, monoclonal antibodies are prepared using hybridoma technologyby fusing antibody producing B cells from immunized animals with myelomacells and selecting the resulting hybridoma cell line producing thedesired antibody. Cells such as COS-7 cells, LM(tk-) cells, NIH-3T3cells or 293 human embryonic cells comprising DNA encoding the Y2receptor and thereby expressing the Y2 receptor may be used asimmunogens to raise such an antibody. Alternatively, synthetic peptidesmay be prepared using commercially available machines and the amino acidsequences shown in FIGS. 2, 8, and 9 (Seq. I.D. Nos. 2, 4, and 6,respectively). As a still further alternative, DNA, such as a cDNA or afragment thereof, may be cloned and expressed and the resultingpolypeptide recovered and used as an immunogen. These antibodies areuseful to detect the presence of Y2 receptors encoded by the isolatedDNA, or to inhibit the function of the receptors in living animals, inhumans, or in biological tissues or fluids isolated from animals orhumans.

This invention provides a pharmaceutical composition which comprises anamount of an antibody directed to a Y2 receptor effective to blockbinding of ligands to the Y2 receptor, and a pharmaceutically acceptablecarrier. A monoclonal antibody directed to an epitope of a Y2 receptorpresent on the surface of a cell and having an amino acid sequencesubstantially the same as an amino acid sequence for a cell surfaceepitope of the Y2 receptor included in the amino acid sequences shown inFIGS. 2, 8 and 9 are useful for this purpose.

This invention also provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan amount of the pharmaceutical composition described above effective toblock binding of ligands to the Y2 receptor, thereby treating theabnormality. In a one embodiment, the Y2 receptor is a human Y2receptor. In another embodiment, the Y2 receptor is a rat Y2 receptor.Binding of the antibody to the receptor prevents the receptor fromfunctioning, thereby neutralizing the effects of activity of thereceptor. The monoclonal antibodies described above are both useful forthis purpose. Some examples of such abnormalities are hypertension,gastrointestinal disorders, epilepsy, sleep disorders, and cognitivedisorders (58-72).

This invention provides a method of detecting the presence of a Y2receptor on the surface of a cell which comprises contacting the cellwith an antibody directed to the Y2 receptor, under conditionspermitting binding of the antibody to the receptor, and detecting thepresence of the antibody bound to the cell, thereby detecting thepresence of a Y2 receptor on the surface of the cell. Such a method isuseful for determining whether a given cell is defective in expressionof Y2 receptors on the surface of the cell. Bound antibodies aredetected by methods well known in the art, for example by bindingfluorescent markers to the antibodies and examining the cell sampleunder a fluorescence microscope to detect fluorescence on a cellindicative of antibody binding. The monoclonal antibodies describedabove are useful for this purpose.

This invention provides a transgenic nonhuman mammal expressing nucleicacid encoding a Y2 receptor. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. This invention also provides a transgenic nonhuman mammalcomprising a homologous recombination knockout of the native Y2receptor. This invention also provides a transgenic nonhuman mammalwhose genome comprises antisense nucleic acid complementary to nucleicacid encoding a Y2 receptor so placed as to be transcribed intoantisense mRNA which is complementary to mRNA encoding a Y2 receptor andwhich hybridizes to mRNA encoding a Y2 receptor thereby reducing itstranslation.

The nucleic acid may additionally comprise an inducible promoter oradditionally comprise tissue specific regulatory elements, so thatexpression can be induced, or restricted to specific cell types.Examples of nucleic acid are DNA or cDNA molecules having a codingsequence substantially the same as the coding sequences shown in FIGS.1, 8, and 9. An example of a transgenic animal is a transgenic mouse.Examples of tissue specificity-determining regions are themetallothionein promotor (77) and the L7 promotor (78).

Animal model systems which elucidate the physiological and behavioralroles of Y2 receptors are produced by creating transgenic animals inwhich the activity of a Y2 receptor is either increased or decreased, orthe amino acid sequence of the expressed Y2 receptor protein is altered,by a variety of techniques. Examples of these techniques include: 1)Insertion of normal or mutant versions of nucleic acid encoding a Y2receptor or homologous animal versions of these genes, bymicroinjection, retroviral infection or other means well known to thoseskilled in the art, into appropriate fertilized embryos in order toproduce a transgenic animal (79). 2) Homologous recombination (80, 81)of mutant or normal, human or animal versions of these genes with thenative gene locus in transgenic animals to alter the regulation ofexpression or the structure of these Y2 receptors. The technique ofhomologous recombination is well known in the art. It replaces thenative gene with the inserted gene and so is useful for producing ananimal that cannot express native receptor but does express, forexample, an inserted mutant receptor, which has replaced the nativereceptor in the animal's genome by recombination, resulting inunderexpression of the receptor. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added receptors, resulting inoverexpression of the receptor. One means available for producing atransgenic animal, with a mouse as an example, is as follows: Femalemice are mated, and the resulting fertilized eggs are dissected out oftheir oviducts. The eggs are stored in an appropriate medium such as M2medium (79). DNA or cDNA encoding a Y2 receptor is purified from avector (such as plasmid pcEXV-hY2, pcEXV-rY2a or pcEXV-rY2b describedabove) by methods well known in the art. Inducible promoters may befused with the coding region of the nucleic acid to provide anexperimental means to regulate expression of the trans-gene.Alternatively, or in addition, tissue specific regulatory elements maybe fused with the coding region to permit tissue-specific expression ofthe trans-gene. The nucleic acid, in an appropriately buffered solution,is put into a microinjection needle (which may be made from capillarytubing using a pipet puller) and the egg to be injected is put in adepression slide. The needle is inserted into the pronucleus of the egg,and the nucleic acid solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting nucleic acid into the egg cell, and is used hereonly for exemplary purposes.

Since the normal action of receptor-specific drugs is to activate or toinhibit the receptor, the transgenic animal model systems describedabove are useful for testing the biological activity of drugs directedagainst these Y2 receptors even before such drugs become available.These animal model systems are useful for predicting or evaluatingpossible therapeutic applications of drugs which activate or inhibitthese Y2 receptors by inducing or inhibiting expression of the native ortrans-gene and thus increasing or decreasing activity of normal ormutant Y2 receptors in the living animal. Thus, a model system isproduced in which the biological activity of drugs directed againstthese Y2 receptors are evaluated before such drugs become available. Thetransgenic animals which over or under produce the Y2 receptor indicateby their physiological state whether over or under production of the Y2receptor is therapeutically useful. It is therefore useful to evaluatedrug action based on the transgenic model system. One use is based onthe fact that it is well known in the art that a drug such as anantidepressant acts by blocking neurotransmitter uptake, and therebyincreases the amount of neurotransmitter in the synaptic cleft. Thephysiological result of this action is to stimulate the production ofless receptor by the affected cells, leading eventually to decreasedactivity. Therefore, an animal which has decreased receptor activity isuseful as a test system to investigate whether the actions of such drugswhich result in decreased activity are in fact therapeutic. Another useis that if increased activity is found to lead to abnormalities, then adrug which down-regulates or acts as an antagonist to a Y2 receptor isindicated as worth developing, and if a promising therapeuticapplication is uncovered by these animal model systems, activation orinhibition of the Y2 receptor is achieved therapeutically either byproducing agonist or antagonist drugs directed against these Y2receptors or by any method which increases or decreases the activity ofthese Y2 receptors in humans or other mammals.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a transgenic nonhuman animal whose levels of Y2 receptorexpression are varied by use of an inducible promoter which regulates Y2receptor expression. This invention also provides a method ofdetermining the physiological effects of expressing varying levels of Y2receptors which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of Y2 receptor. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. Such animals may beproduced by introducing different amounts of nucleic acid encoding a Y2receptor into the oocytes from which the transgenic animals aredeveloped.

This invention also provides a method for identifying a Y2 receptorantagonist capable of alleviating an abnormality is a subject, whereinthe abnormality is alleviated by decreasing the acitivity of a Y2receptor which comprises administering the antagonist to a transgenicnonhuman mammal described above and determining whether the antagonistalleviates the physical and behavioral abnormalities displayed by thetransgenic nonhuman mammal as a result of the activity of a Y2 receptor,thereby identifying a Y2 antagonist. In one embodiment, the Y2 receptoris a human Y2 receptor, In another embodiment, the Y2 receptor is a ratY2 receptor. This invention further provides an antagonist identified bythe method described above. Examples of nucleic acid molecules are DNAor cDNA molecules having a coding sequence substantially the same as thecoding sequences shown in FIGS. 1, 8, and 9.

This invention provides a pharmaceutical composition comprising anamount of the antagonist described supra effective to alleviate anabnormality wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor and a pharmaceutically acceptable carrier.

This invention further provides a method for treating an abnormality ina subject wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition described above,thereby treating the abnormality.

This invention provides a method for identifying a Y2 receptor agonistcapable of alleviating an abnormality wherein the abnormality isalleviated by activation of a Y2 receptor which comprises administeringthe agonist to the transgenic nonhuman mammal described above anddetermining whether the agonist alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, therebyidentifying a Y2 receptor agonist. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. This invention further provides an agonist identified by themethod described above.

This invention also provides a pharmaceutical composition comprising aneffective amount of a Y2 receptor agonist identified by the methoddescribed above and a pharmaceutically acceptable carrier.

This invention further provides a method for treating an abnormality ina subject wherein the abnormality is alleviated by activation of a Y2receptor which comprises administering to the subject an effectiveamount of the pharmaceutical composition described above, therebytreating the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the expression of a specific Y2 receptor allelewhich comprises: a) obtaining nucleic acid of subjects suffering fromthe disorder; b) performing a restriction digest of the nucleic acidwith a panel of restriction enzymes; c) electrophoretically separatingthe resulting nucleic acid fragments on a sizing gel; d) contacting theresulting gel with a nucleic acid probe capable of specificallyhybridizing to nucleic acid encoding a Y2 receptor and labelled with adetectable marker; e) detecting labelled bands which have hybridized tothe nucleic acid encoding a Y2 receptor labelled with a detectablemarker to create a unique band pattern specific to the nucleic acid ofsubjects suffering from the disorder; f) preparing nucleic acid obtainedfor diagnosis by steps a-e; and g) comparing the unique band patternspecific to the nucleic acid of subjects suffering from the disorderfrom step e and the nucleic acid obtained for diagnosis from step f todetermine whether the patterns are the same or different and thereby todiagnose predisposition to the disorder if the patterns are the same.This method may also be used to diagnose a disorder associated with theexpression of a specific Y2 receptor allele. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor.

This invention provides a method of preparing the isolated, purified Y2receptor which comprises a) constructing a vector adapted for expressionin a cell which comprises the regulatory elements necessary for theexpression of nucleic acid in the cell operatively linked to the nucleicacid encoding a Y2 receptor as to permit expression thereof, wherein thecell is selected form the group consisting of bacterial cells, yeastcells, insect cells and mammalian cells; b) inserting the vector of step(a) in a suitable host cell; c) incubating the cells of step (b) underconditions allowing the expression of a Y2 receptor; d) recovering thereceptor so produced; and e) purifying the receptor so recovered. Anexample of an isolated Y2 receptor is an isolated protein havingsubstantially the same amino acid sequence as the amino acid sequencesshown in FIGS. 2, 8 and 9. For example, cells can be induced to expressreceptors by exposure to substances such as hormones. The cells can thenbe homogenized and the receptor isolated from the homogenate using anaffinity column comprising, for example, PYY or NPY or another substancewhich is known to bind to the receptor. The resulting fractions can thenbe purified by contacting them with an ion exchange column, anddetermining which fraction contains receptor activity or bindsanti-receptor antibodies.

The above described method for preparing a Y2 receptor uses recombinantDNA technology methods well known in the art. For example, isolatednucleic acid encoding Y2 receptor is inserted in a suitable vector, suchas an expression vector. A suitable host cell, such as a bacterial cell,or a eukaryotic cell such as a yeast cell, is transfected with thevector. Y2 receptor is isolated from the culture medium by affinitypurification or by chromatography or by other methods well known in theart.

This invention identifies for the first time a new receptor protein, itsamino acid sequence, its human gene and its rat homologue. Furthermore,this invention describes a previously unrecognized group of receptorswithin the definition of a Y2 receptor. The information and experimentaltools provided by this discovery are useful to generate new therapeuticagents, and new therapeutic or diagnostic assays for this new receptorprotein, its associated mRNA molecule or its associated genomic DNA. Theinformation and experimental tools provided by this discovery will beuseful to generate new therapeutic agents, and new therapeutic ordiagnostic assays for this new receptor protein, its associated mRNAmolecule, or its associated genomic DNA.

Specifically, this invention relates to the first isolation of a humangenomic clone encoding a Y2 receptor. A new human gene for the receptoridentified herein as Y2 has been identified and characterized. Inaddition, the human Y2 receptor has been expressed in 293 humanembryonic kidney cells. The pharmacological binding properties of theprotein encoded have been determined, and these binding propertiesclassify this protein as a novel human NPY/PYY receptor which wedesignate as a human Y2 receptor. Mammalian cell lines expressing thishuman Y2 receptor at the cell surface have been constructed, thusestablishing the first well-defined, cultured cell lines with which tostudy this Y2 receptor.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

cDNA Cloning

Total RNA was prepared by a modification of the guanidine thiocyanatemethod (13), from 6 grams of human hippocampus. Poly A⁺ RNA was purifiedwith a FastTrack kit (Invitrogen Corp., San Diego, Calif.). Doublestranded (ds) cDNA was synthesized from 4 μg of poly A⁺ RNA according toGubler and Hoffman (14), except that ligase was omitted in the secondstrand cDNA synthesis. The resulting DS cDNA was ligated to BstxI/EcoRIadaptors (Invitrogen Corp.), the excess of adaptors was removed bychromatography on Sephacryl 500 HR (Pharmacia-LKB) and the ds-cDNA sizeselected by chromatography on Sephacryl 1000 (Pharmacia-LKB). Highmolecular weight fractions were ligated in pcEXV.BS (An Okayama and Bergexpression vector) cut by BstxI as described by Aruffo and Seed (15).The ligated DNA was electroporated in E. coli MC 1061 (Gene Pulser,Biorad). A total of 2.2×10⁶ independent clones with an insert mean sizeof 3 kb could be generated. The library was plated on Petri dishes(Ampicillin selection) in pools of 0.4 to 1.2×10⁴ independent clones.After 18 hours amplification, the bacteria from each pool were scraped,resuspended in 4 mL of LB media and 1.5 mL processed for plasmidpurification by the alkali method (16). 1 mL aliquots of each bacterialpool were stored at -85° C. in 20% glycerol.

Isolation of a cDNA Clone Encoding a Human Hippocampal Y2 Receptor

DNA from pools of ≈5000 independent clones was transfected into COS-7cells by a modification of the DEAE-dextran procedure (17). COS-7 cellswere grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal calf serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin,2mM L-glutamine (DMEM-C) at 37° C. in 5% CO₂. The cells were seeded oneday before transfection at a density of 30,000 cells/cm² in 6 wellplates (Becton Dickinson, Lincoln Park, N.J.). On the next day, cellswere washed twice with Phosphate Buffer Saline (PBS), 400 μl oftransfection cocktail was added containing 1/10 of the DNA from eachpool and DEAE-dextran (500 μg/mL) in PBS. After a 30 min. incubation at37° C., 1.6 mL of chloroquine (80 μM in DMEM-C) was added and the cellsincubated a further 2.5 hours at 37° C. The media was aspirated fromeach well and 1 mL of 10% DMSO in DMEM-C added. After 2.5 min.incubation at room temperature, the media was aspirated, each wellwashed once with 1 mL PBS and the cells incubated 24 hours in DMEM-C.The cells were then trypsinized and seeded on Lab-Tek chamber slides (1chamber, Permanox slide from Nunc Inc., Naperville, Ill.), incubated in2 ml DMEM-C for another 24 hours and the binding assay was performed onthe slides.

After two washes with PBS, positive pools were identified by incubatingthe cells with 1 nM (3×10⁶ cpm per slide) of porcine [¹²⁵ I]-PYY (NewEngland Nuclear; specific activity=2200 Ci/mmol) in 20 mM Hepes-NaOH pH7.4, CaCl₂ 1.26 mM, MgSO₄ 0.81 mM, KH₂ PO₄ 0.44 mM, KCl 5.4, NaCl 10 mM,0.1% bovine serum albumin, 0.1% bacitracin for 1 hour at roomtemperature. After six washes (five seconds each) in binding bufferwithout ligand, the monolayers were fixed in 2.5% glutaraldehyde in PBSfor five minutes, washed twice two minutes in PBS, dehydrated in ethanolbaths for two minutes each (70, 80, 95, 100%) and air dried.

The slides were then dipped in 100% photoemulsion (Kodak type NTB2) at42° C. and exposed in the dark for 48 hours at 4° C. in light proofboxes containing drierite. Slides were developed for three minutes inKodak D19 developer (32 g/l of water), rinsed in water, fixed in Kodakfixer for 5 minutes, rinsed in water, air dried and mounted withAqua-Mount (Lerner Laboratories, Pittsburgh, Pa.). Slides were screenedat 25×total magnification.

A single clone, CG-13, was isolated by sib selection as described (18).DS-DNA was sequenced with a Sequenase kit (US Biochemical, Cleveland,Ohio) according to the manufacturer. Nucleotide and peptide sequencesanalysis were performed with GCG programs (Genetics Computer group,Madison, Wis.).

Northern Blot

A multiple tissue Northern blot (MTN blot, Contech, Palo Alto, Calif.)carrying mRNA purified from various human brain areas was hybridized athigh stringency according to the manufacturer's specifications. Theprobe was a 1.15 kb DNA fragment corresponding to the entire codingregion of the human Y2 receptor as shown in FIG. 10.

Southern Blot

A Southern blot (Geno-Blot, Clontech, Palo Alto, Calif.) containinghuman genomic DNA cut with five different enzymes (8 μg DNA per lane)was hybridized at high stringency according to the manufacturer'sspecifications. The probe was a DNA fragment corresponding to theTM1-TM5 coding region of the human Y2 receptor, as shown in FIG. 11.

Cloning and Expression of Two Isoforms of the Rat NPY/PYY (Y2) Receptor

To obtain the rat homologue of the human NPY/PYY (Y2) receptor, wedesigned and synthesized oligonucleotide probes derived from thenucleotide sequences corresponding approximately to the transmembrane(TM) regions of the amino acid sequence of the human Y2 receptor (TM1-7)as shown in FIG. 11. The overlapping oligomers used were as follows:

(TM1: nts. #190-257, (+)strand/5'-CAAGTTGTTCTCATATTGGCCTACTGCTCCATCATCTTGCTTGGGGTAAT-3' (Seq.I.D. No. 7) and (-)strand/5'-ATCACCACATGGATCACCAAGGAGTTGCCAATTACCCCAAGCAAGATGAT-3' (Seq.I.D. No. 8)

TM2: nts. #301-370, (+)strand/5'-TTTTTCATTGCCAATCTGGCTGTGGCAGATCTTTTGGTGAACACT-3' (Seq. I.D.No. 9) and (-)strand/5'-AGGTAAGAGTGAACGGTAGACACAGAGTGTTCACCAAAAGATCTG-3' (Seq. I.D.No. 10).

TM3: nts. #411-480, (+)strand/5'-CCACCTGGTGCCCTATGCCCAGGGCCTGGCAGTACAAGTATCCAC-3' (Seq. I.D.No. 11) and (-)strand/5'-CAGGGCAATTACTGTCAAGGTGATTGTGGATACTTGTACTGCCAG-3' (Seq. I.D.No. 12).

TM4: nts. #531-600, (+)strand/5'-AATCAGCTTCCTGATTATTGGCTTGGCCTGGGGCATCAGTGCCCT-3' (Seq. I.D.No. 13) and (-)strand/5'-GAAGATGGCCAGGGGACTTGCCAGCAGGGCACTGATGCCCCAGGC-3' (Seq. I.D.No. 14)

TM5: nts. #691-760, (+)strand/5'-ACTGTCTATAGTCTTTCTTCCTTGTTGATCTTGTATGTTTTGCCT-3' (Seq. I.D.No. 15) and (-)strand/5'-TGTAGGAAAATGATATAATGCCCAGAGGCAAAACATACAAGATCA-3' (Seq. I.D.NO. 16)

TM6: nts. #850-919, (+)strand/5'-CTGGTGTGTGTGGTGGTGGTGTTTGCGGTCAGCTGGCTGCCTCTC-3' (Seq. I.D.No. 17) and (-)strand/5'-TGTCAACGGCAAGCTGGAAGGCATGGAGAGGCAGCCAGCTGACCG-3' (Seq. I.D.No. 18)

TM7: NTS. #955-1028, (+)strand/5'-CTCATCTTCACAGTGTTCCACATCATCGCCATGTGCTCCACTTTTGC-3' (Seq. I.D.No. 19) and (-)strand/5'-TTCATCCAGCCATAGAGAAGGGGATTGGCAAAAGTGGAGCACATGGC-3' (Seq. I.D.No. 20).

The probes were labeled with [³² P]-ATP and [³² p]-CTP by synthesis withthe large fragment of DNA polymerase.

Hybridization was performed at 40° C. in a solution containing 25%formamide, 10% dextran sulfate, 5× SSC (1× SSC is 0.15 M sodiumchloride, 0.015 M sodium citrate), 1× Denhardt's (0.02%polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and100 μg/ml of sonicated salmon sperm DNA. The filters were washed at 40°C. in 0.1× SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposedat -70° C. to Kodak XAR film in the presence of one intensifying screen.Lambda phage hybridizing to the probes were plaque purified bysuccessive plating and rescreening. A genomic clone hybridizing with sixout of seven TM probes, designated rs5a, was isolated using this method.A 4.0 kb EcoRI fragment of rs5a was subcloned into the eukaryoticexpression vector EXJ.RH modified from pcEXV-3 (73) for sequenceanalysis and expression studies. The nucleotide sequence of the fragmentin EXJ.RH was analyzed on both strands by the Sanger dideoxy nucleotidechain-termination method (82) using Sequenase (U.S. Biochemical Corp.,Cleveland, Ohio).

A second genomic clone, termed rs26a, was also isolated using thehybridization conditions described above and exhibited the samehybridization profile with TM probes. In contrast with rs5a, however,rs26a contained an internal EcoRI restriction enzyme site not present inthe other clone. To further investigate potential differences betweenthe two clones, a 3.9 kb SalI/KpnI fragment of rs26a was subcloned intothe expression vector EXJ.HR for sequence analysis and expressionstudies. The nucleotide sequence of the fragment was analyzed on bothstrands by the Sanger dideoxy nucleotide chain-termination method asdescribed above.

Cell Culture

COS-7 cells were grown on 150 mm plates in Dulbecco's Modified EagleMedium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine,100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stockplates of COS-7 cells were trypsinized and split 1:6 every 3-4 days.

Human embryonic kidney cells 293 cells were grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of 293 cells weretrypsinized and split 1:6 every 3-4 days.

Mouse embryonic fibroblast NIH-3T3 cells were grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells weretrypsinized and split 1:15 every 3-4 days.

SK-N-Be(2) human neuroblastoma cells were grown similarly in 225 cm²flasks using 50% Eagle's Modified Essential Media, 50% Ham's NutrientMixture F-12, 15% fetal bovine serum, 2 mM glutamine, 100 units/mlpenicillin/80 units/ml streptomycin, and 1% non-essential amino acids.Stock flasks of SK-N-Be(2) cells were trypsinized and split 1:10 every 7days.

DNA Transfection for Pharmacological Characterization

All cloned receptor subtypes studied (human Y1 , human Y2, human Y4, ratY2a and rat Y2b) were transiently transfected into COS-7 cells by theDEAE-dextran method, using 1 μg of DNA/10⁶ cells (17). The cDNAcorresponding to the cloned Y4 receptor was disclosed in U.S. patentapplication Ser. No. 08/176,412 filed on Dec. 28, 1993, now U.S. Pat.No. 5,516,653.

Membrane Preparation

Membranes were harvested from COS-7 cells 48 hours after transfectionand from SK-N-Be(2) seven days after splitting. Adherent cells werewashed twice in ice-cold phosphate buffered saline (138 mM NaCl, 8.1 mMNa₂ HPO₄, 2.5 mM KCl, 1.2 mM KH2PO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 7.4)and lysed by sonication in ice-cold hypotonic buffer (20 mM Tris-HCl, 5mM EDTA, pH 7.7). Large particles and debris were cleared by low speedcentrifugation (200×g, 20 min, 4° C.). Membranes were collected from thesupernatant fraction by high speed centrifugation (32,000×g, 18 min, 4°C.), washed with ice-cold hypotonic buffer, and collected again by highspeed centrifugation (32,000×g, 18 min, 4° C.). The final membranepellet was resuspended by sonication into a small volume (˜500 μl) ofice-cold binding buffer (10 mM NaCl, 20 mM HEPES, 0.22 mM KH₂ PO₄, 1.26mM CaCl₂, 0.81 mM MgSO₄, pH 7.4). Protein concentration was measured bythe Bradford method (19) using Bio-Rad Reagent, with bovine serumalbumin as a standard.

Radioligand Binding to Membrane Suspensions

Membrane suspensions were diluted in binding buffer supplemented with0.1% bovine serum albumin and 0.1% bacitracin to yield membrane proteinconcentrations of ˜0.02 mg/ml for human Y1 receptors, ˜0.003 mg/ml forCG-13 receptors, and ˜0.25 mg/ml for SK-N-Be(2) (under these assayconditions, non-specific binding of ¹²⁵ I-PYY to membranes was less than10%). ¹²⁵ I-PYY and non-labeled peptide competitors were also diluted todesired concentrations in supplemented binding buffer. Individualsamples were then prepared in 96-well polypropylene microliter plates bymixing membrane suspensions (200 ul), ¹²⁵ I-PYY (25 ul), and non-labeledpeptides or supplemented binding buffer (25 ul). Samples were incubatedin a 30° C. water bath with constant shaking for 120 min. Incubationswere terminated by filtration over Whatman GF/C filters (pre-coated with0.5% polyethyleneimine and air-dried before use). Filter-trappedmembranes were counted for ¹²⁵ I in a gamma counter. Non-specificbinding was defined by 100 nM human NPY. Specific binding in time courseand competition studies was typically 80%; most non-specific binding wasassociated with the filter. Binding data were analyzed using nonlinearregression and statistical techniques available in the GraphPAD InPlotpackage (San Diego, Calif.).

Creation of a Stably Expressing Cell Line

pcEXV-hY2 DNA was transfected into the 293 human embryonic kidney cellline by the calcium phosphate transfection method. The 293 cells weregrown in minimal essential medium (MEM) with Hank's salts, plus 2 mMglutamine, 100 international units of penicillin, streptomycin at 100ug/ml, and 10% fetal calf serum, in 5% CO₂ at 37° C. Stably transfectedcells were selected for two weeks in media containing G-148 (1 mg/ml)and screened for the ability to bind ¹²⁵ I-PYY. Several clones wereselected based on preliminary measurements of cell density. One positiveclone, designated 293-hY2-10, was chosen for further characterization inbinding and functional assays. This clone displayed saturable binding of¹²⁵ I-porcine PYY in membrane preparations: B_(max) =880 fmol/mgmembrane protein, K_(d) =3 pM, (n=3). When incubated with variousconcentrations of human PYY, it elicited a concentration-dependentinhibition of forskolin-stimulated cAMP accumulation as determined byradioimmunoassay. Clone 293-hY2-10 also elicited aconcentration-dependent increase in free intracellular calcium asdetermined by Fura-2 florescence. The calcium response, which probablyreflects mobilization of intracellular calcium stores, was inhibited bypretreatment of cells with pertussis toxin. EC₅₀ values for both thecAMP and the calcium response are currently under investigation.pcEXV-hY2 DNA was also transfected into the mouse embryonic NIH-3T3 cellline using the methods described above to create another cell linestably expressing human Y2 receptors. A clone designated N-hY2-5 wasselected and characterized as above.

Tissue Localization and Gene Expression: Reverse Transcriptase PCR

Human tissues obtained from National Disease Research Interchange werehomogenized and total RNA extracted using guanidine isothiocyanate/CsClcushion method. RNA was treated with DNase to remove any contaminatinggenomic DNA. cDNA was prepared from total RNA with random hexanucleotideprimers using the reverse transcriptase Superscript II (BRL,Gaithersburg, Md.). An aliquot of the first strand cDNA (250 ng of totalRNA) was amplified in a 50 μl PCR reaction mixture (200 μM dNTPs finalconcentration) containing 1.2 U of Taq polymerase in the buffer suppliedby the manufacturer (Perkin-Elmer Corporation), and 1 μM of primers,using a program consisting of 30 cycles of 94° C./2', 68° C./2', and 72°C./3', with a pre- and post-incubation of 95° C./5' and 72° C./10',respectively. PCR primers for human Y2 were designed against the humanY2 sequence in the third intracellular loop and carboxyl terminalregions: 5'-GGGAGTATTCGCTGATTGAGATCAT-3' (SEQ. I.D. No. 21) and5'-GCCTTGAATGTCACGGACACCTC-3' (SEQ. I.D. No. 22), respectively.

The PCR products were run on a 1.5% agarose gel and transferred tocharged nylon membranes (Zetaprobe GT, BioRad), and analyzed as Southernblots. Hybridization probes corresponding to the receptor region flankedby PCR primers were prepared(5'-CTGATGGTAGTGGTCATTTGCAGCTCCAGGACTGACATGGTTCTT-3') (SEQ. I.D. No. 23)and pre-screened for the absence of cross-reactivity with human Y1 andY4 receptor subtypes.

Filters were hybridized with the phosphorylated probes and washed underhigh stringency. Labeled PCR products were visualized on X-ray film.Similar PCR and Southern blot analyses were conducted with primers andprobe directed to the housekeeping gene, glyceraldehyde-3-phosphatedehydrogenase (Clontech, Palo Alto, Calif.), and demonstrated that equalamounts of cDNA from the different tissues were being assayed for Y2receptor expression.

Localization of NPY Y2 Messenger RNA in the Rat Central Nervous System

The distribution of NPY Y2 mRNA in the rat brain was determined using insitu hybridization histochemistry. Male Sprague-Dawley rats wereeuthanized with CO₂, decapitated and the brains rapidly removed andfrozen in isopentane. Coronal sections were cut at 11 μm on a cryostatand thaw-mounted onto poly-L-lysine coated slides and stored at -80° C.until use. Prior to hybridization, tissues were fixed in 4%paraformaldehyde, treated with 5 mM dithiothreitol, acetylated in 0.1 Mtriethanolamine containing 0.25% acetic anhydride, delipidated withchloroform, and dehydrated in graded ethanols.

The oligonucleotide probes employed to characterize the distribution ofthe NPY Y2 mRNA were synthesized using a Cyclone Plus DNA synthesizer(Milligen/Biosearch) and gel-purified. The probes used and theirsequences are given in Table 7. Probe specificity was established byperforming the in situ hybridization protocol described below on cellstransfected with the rat NPY Y2 DNA (supra), or on nontransfectedcontrol cells. In addition, both sense and antisense probes wereemployed on cells and rat tissues.

Probes were 3'-end labeled with ³⁵ S-dATP (1200 Ci/mmol, New EnglandNuclear, Boston, Mass.) to a specific activity of 10⁹ dpm/μg usingterminal deoxynucleotidyl transferase (Boehringer Mannheim;Indianapolis, Ind.). The radiolabeled probes were purified on Biospin 6chromatography columns (Bio-Rad; Richmond, Calif.), and diluted inhybridization buffer to a concentration of 1.5×10⁴ cpm/μl. Thehybridization buffer consisted of 50% formamide, 4× sodium citratebuffer (1× SSC=0.15 M NaCl and 0.015 M sodium citrate), 1× Denhardt'ssolution (0.2% polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serumalbumin), 50 mM dithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/mlyeast tRNA, and 10% dextran sulfate. One hundred μl of the diluted probewas applied to each section, which was then covered with a Parafilmcoverslip. Hybridization was carried out overnight in humid chambers at40 to 55° C. The following day the sections were washed in two changesof 2× SSC for one hour at room temperature, in 0.1× SSC for 30 min at50-60° C., and finally in 0.1× SSC for 30 min at room temperature.Tissues were dehydrated in graded ethanols and apposed to Kodak XAR-5film for 3 days to 6 weeks at -20° C., then dipped in Kodak NTB3autoradiography emulsion diluted 1:1 with 0.2% glycerol water. Afterexposure at 4° C. for 2 to 8 weeks, the slides were developed in KodakD-19 developer, fixed, and counterstained with hematoxylin and eosin.

Functional Assay: Radioimmunoassay of cAMP

Stably transfected cells were seeded into 96-well microliter plates andcultured until confluent. To reduce the potential for receptordesensitization, the serum component of the media was reduced to 1.5%for 4 to 16 hours before the assay. Cells were washed in Hank's bufferedsaline, or HBS (150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl, 1 mMMgCl₂, and 10 mM glucose) supplemented with 0.1% bovine serum albuminplus 5 mM theophylline and pre-equilibrated in the same solution for 20min at 37° C. in 5% CO₂. Cells were then incubated 5 min with 10 μMforskolin and various concentrations of receptor-selective ligands. Theassay was terminated by the removal of HBS and acidification of thecells with 100 mM HCl. Intracellular cAMP was extracted and quantifiedwith a modified version of a magnetic bead-based radioimmunoassay(Advanced Magnetics, Cambridge, Mass.). The final antigen/antibodycomplex was separated from free ¹²⁵ I-cAMP by vacuum filtration througha PVDF filter in a microliter plate (Millipore, Bedford, Mass.). Filterswere punched and counted for ¹²⁵ I in a Packard gamma counter. Bindingdata were analyzed using nonlinear regression and statistical techniquesavailable in the GraphPAD Prism package (San Diego, Calif.).

Functional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM.Stably transfected cells were seeded onto a 35 mm culture dishcontaining a glass coverslip insert. Cells were washed with HBS and thenloaded with 100 μl of Fura-2/AM (10 μM) for 20 to 40 min. After washingwith HBS to remove the Fura-2/AM solution, cells were equilibrated inHBS for 10 to 20 min. Cells were then visualized under the 40× objectiveof a Leitz Fluovert FS microscope and fluorescence emission wasdetermined at 510 nM with excitation wave lengths alternating between340 nM and 380 nM. Raw fluorescence data were converted to calciumconcentrations using standard calcium concentration curves and softwareanalysis techniques.

Reagents

Cell culture media and supplements were from Specialty Media(Lavallette, N.J.). Cell culture plates (150 mm) were from Corning(Corning, N.Y.). Cell culture flasks (225 cm²) and polypropylenemicroliter plates were from Co-star (Cambridge, Mass.). Porcine ¹²⁵I-PYY was from New England Nuclear (Boston, Mass.). NPY and relatedpeptide analogs were from either Bachem California (Torrance, Calif.) orPeninsula (Belmont, Calif.). Whatman GF/C filters were Brandel(Gaithersburg, Md.). Bio-Rad Reagent was from Bio-Rad (Hercules,Calif.). Bovine serum albumin and bacitracin were from Sigma (St. Louis.Mo.). All other materials were reagent grade.

RESULTS

Isolation of a cDNA Clone Encoding a Human Hippocampal Y2 Receptor

In order to clone a human NPY receptor subtype (Y2), we used anexpression cloning strategy in COS-7 cells (20, 21, 22). This strategywas chosen for its extreme sensitivity since it allows detection of asingle "receptor positive" cell by direct microscopic autoradiography.

Since the Y2 receptor is described as a presynaptic receptor, it isdifficult to locate cell bodies that actually contain this specific mRNAin restricted brain areas. We reasoned that human hippocampus was a goodsource of mRNA since it contains both a large number of interneurons andhas been shown to carry a particularly dense population of Y2 receptors(23, 24, 25, 26). A human hippocampal cDNA library of 2.2×10⁶independent recombinants with a 3 kb average insert size wasfractionated into 440 pools of ≈5000 independent clones. From the first200 pools tested, three gave rise to positive cells in the screeningassay (#145, 158 and 189). The last 220 pools tested were all negative.

Since both Y1 and Y2 receptor subtypes are expressed in the hippocampus(2), we analyzed the DNA of positive pools by PCR with Y1 specificprimers. Pools #145 and #158 turned out to contain cDNAs encoding an Y1receptor, but pool #189, negative by PCR (data not shown), likelycontained a cDNA encoding a human hippocampal NPY receptor that was notY1. Pool #189 was subdivided in 20 pools of 1000 clones each, and apreliminary pharmacological characterization was run on COS-7 cellstransfected with DNA prepared from the secondary pools. This preliminaryanalysis revealed that a 100 fold excess of cold [Leu³¹ -Pro³⁴ ]NPYtotally inhibited binding of ¹²⁵ I-PYY to control COS-7 cellstransfected with the Y1 gene. In contrast, no significant inhibition ofbinding was observed when the same experiment was performed on COS-7cells transfected with secondary pool #189-17 (data not shown). This isconsistent with pool #189 containing a cDNA encoding a human hippocampalY2 receptor. The sib selection was therefore pursued on pool #189 untila single clone was isolated (designated CG-13).

The isolated clone carries a 4.2 kb cDNA. This cDNA contains an openreading frame between nucleotides 1002 and 2147 that encodes a 381 aminoacid protein (SEQ. I.D. No. 2). The unusually long 5' untranslatedregion could be involved in the regulation of translation efficiency ormRNA stability. The flanking sequence around the putative initiationcodon conforms to the Kozak consensus sequence for optimal translationinitiation (27, 28).

The hydrophobicity plot displayed seven hydrophobic, putative membranespanning regions which makes the human hippocampal Y2 receptor a memberof the G-protein coupled superfamily. The nucleotide and deduced aminoacid sequences are shown in FIG. 1 and FIG. 2, respectively.

Like most G-protein coupled receptors, the Y2 receptor contains aconsensus sequence for N-linked glycosylation, in the amino terminus(position 11) involved in the proper expression of membrane proteins(29). The Y2 receptor carries two highly conserved cysteine residues inthe first two extracellular loops that are believed to form a disulfidebond stabilizing the functional protein structure (30). The Y2 receptorshows 7 potential phosphorylation sites for protein kinase C inpositions 11, 27, 64, 145, 188, 250 and 340, 2 casein kinase sites inpositions 174 and 358, and 2 cAMP- and cGMP-dependent protein kinasephosphorylation sites in positions 146 and 350. It should be noted that7 of those 11 potential phosphorylation sites are located inintra-cellular loops 1, 2 and 3 as well as in the carboxyl terminus ofthe receptor and therefore could play a role in regulating functionalcharacteristics of the Y2 receptor (30). A potential palmitoylation siteis present in the sequence at the cysteine found in position 326. Alarge number of G-protein coupled receptors carry a cysteine in the sameposition and O'Dowd et al. have speculated that it plays an importantrole in the functional coupling of the human β₂ -adrenergic receptor(31). The formation of this additional cytosolic loop may influence themobility of the receptor across the membrane (32).

When compared to the published human Y1 cDNA clone (10, 11) the Y2sequence shows surprisingly low homology both at the nucleotide level,48.1% (FIG. 3) and overall amino acid level, 31% (FIG. 4). Thetransmembrane domain identity of the human hippocampal Y2 receptor withother 7 TM receptors is shown in Table 1. The low TM identity with otherG-protein coupled receptor families, with other peptide receptors andespecially with the Y1 subtype raises the possibility that Y2 receptorsubtypes belong to a new distinct sub-family of 7 TM peptide receptors.Conversely, NPY receptor subtypes could form a sub-family where membersshow unusually low levels of overall homology. Applicants have alsocloned the human Y4 receptor, and this receptor also exhibits a lowdegree of homology with the human Y2 receptor (Table 1). It isinteresting to observe that the mouse orphan receptor MUSGIR (mouseglucocorticoid induced receptor, 33) shows the highest TM identity (42%,Table 1) with our human Y2 receptor. The same comparison between humanY1 (or Y4) and Y2 TM regions only gives a score of 41% identity. If wewere comparing the human Y2 receptor sequence with the human homolog ofthe MUSGIR receptor, the level of identity might even be higher.Therefore the MUSGIR receptor could be related to the NPY receptors andbind members of the pancreatic polypeptide ligand family. A fullpharmacological evaluation of the human GIR homolog with NPY, PYY and PPrelated ligands is now underway to verify this hypothesis.

Using the human Y2 probe, northern hybridizations reveal a unique bandat 4.3 kb in human brain after a three-day exposure (FIG. 16). This isin good agreement with the 4.2 kb cDNA that we isolated by expressioncloning and indicates that our cDNA clone is full-length. The mRNAencoding the human Y2 receptor is present in significant amounts inamygdala, corpus callosum, hippocampus, and subthalamic nucleus. A faintband is detectable in caudate nucleus, hypothalamus and substantianigra. No signal could be detected in thalamus. It should be noted thatClontech's MTN blot does not carry any mRNA from cortex or brain stem.

Southern hybridizations to human genomic DNA followed by high stringencywashes (FIG. 17) suggest that the human genome contains a single Y2receptor gene (single band with EcoRI, HindIII, BamHI and PstI). Thefaint bands at 9 and 12 kb observed with BglII can be explained by thepresence of two BglII restriction sites in the coding region of thehuman Y2 sequence and are also consistent with a single Y2 receptorgene.

Pharmacology of the Transiently Expressed Human Y2 Receptor

The Y2-like pharmacology of CG-13, originally identified by whole cellautoradiographic techniques, was further defined by membrane bindingassays. The gene for the human hippocampal Y2 receptor was transientlyexpressed in COS-7 cells for full pharmacological evaluation. ¹²⁵ I-PYYbound specifically to membranes from COS-7 cells transiently transfectedwith the CG-13 construct. The time course of specific binding wasmeasured in the presence of 0.06 nM ¹²⁵ I-PYY (FIG. 5). The associationcurve was biphasic, with approximately 55% of the specific bindingoccurring during an initial rapid phase and 45% following a slower timecourse. For the rapid phase, the observed association constant (K_(obs))was 1.28±0.02 min⁻¹ and t_(1/2) was 0.5 min; equilibrium binding was 95%complete within 2 min and 100% complete within 5 min (n=3). For the slowphase, K_(obs) was 0.02±0.00 min⁻¹ and t_(1/2) was 37 min; equilibriumbinding was 90% complete within 120 min, 95% complete within 160 min and100% complete within 280 min (n=3). Total equilibrium binding, composedof both phases, was 95% complete within 120 min and 100% complete within240 min. The biphasic association curve may reflect a complex pattern ofreceptor surface binding followed by access to deep-seated bindingsites, as has been suggested by Schwartz and co-workers for Y2 receptors(34). For comparison, we also measured the time course of binding tohuman Y1 receptors transiently expressed in COS-7 cells (FIG. 5). Theassociation curve was monophasic, with a K_(obs) of 0.06±0.02 min⁻¹ anda t_(1/2) of 12 min; equilibrium binding was 95% complete within 51 minand 100% complete within 90 min (n=3). The different patterns ofassociation for CG-13 and human Y1 receptors suggest novel mechanisms ofreceptor/ligand interaction.

Saturation binding data for ¹²⁵ I-PYY were fit to a one-site model withan apparent K_(d) of 0.069±0.009 nM and an apparent B_(max) of 7.8±0.4pmol/mg membrane protein, corresponding to approximately 7.5×10⁵receptors/cell (n=3; FIG. 6). Given that the transfection efficiency was20-30% (data not shown), the receptor density on transfected cells wasprobably closer to 3×10⁶ /cell. Membranes from mock-transfected cells,when prepared and analyzed in the same way as those fromCG-13-transfected cells, displayed no specific binding of ¹²⁵ I-PYY. Weconclude that the ¹²⁵ I-PYY binding sites observed under the describedconditions were derived from the CG-13 construct.

Y2 receptor recognition is thought to depend primarily upon the fourC-terminal residues of NPY (Arg³³ -Gln³⁴ -Arg³⁵ -Tyr³⁶ -NH₂) preceded byan amphipathic α-helix (M4, M5); exchange of Gln³⁴ with Pro³⁴ is notwell tolerated (4, 5). We therefore chose several C-terminal fragmentsand C-terminal modified peptides for competition binding studies. Therank order of affinity for selected compounds was derived fromcompetitive displacement of ¹²⁵ I-PYY (FIG. 7 and Table 3). The CG-13receptor was compared with two model systems: 1) the cloned human Y1receptor (10, 11) transiently expressed in COS-7 cells, and 2) theY2-like receptor population expressed by human SK-N-Be(2) neuroblastomacells (2, 8). To our knowledge, no models for human Y3 and human PPreceptors have been described.

CG-13 bound with high affinity to human NPY (K_(i) =0.69 nM) and evenmore so to human PYY (K_(i) =0.39 nM). The K_(i) values are in agreementwith numerous reports of pharmacologically defined Y2 receptors studiedin NPY binding and functional assays (2). The opposite rank order wasobserved with human Y1 receptors, combined with strongerreceptor/binding interactions (K_(i) =0.049 and 0.085 nM for human NPYand human PYY, respectively). It is interesting in this regard thatCG-13 bound ¹²⁵ I-PYY (K_(d) =0.069 nM) with higher affinity than PYY(K_(i) =0.39 nM), suggesting that iodination may stabilize thereceptor/ligand complex. The human Y1 receptor, in contrast, bound both¹²⁵ I-PYY (K_(d) =0.062+0.010 nM, n=3, data not shown) and PYY (K_(i)=0.085 nM) with comparable affinity. The fact that CG-13 and the humanY1 receptor bound NPY, PYY and ¹²⁵ I-PYY with different magnitudes andrank orders of affinity most likely reflects distinct mechanisms ofpeptide recognition which could potentially be exploited for thedevelopment of subtype-selective non-peptide ligands.

CG-13 also bound with high affinity to porcine NPY (K_(i) =0.86 nM),which differs from human NPY by containing Leu¹⁷ in the PP-fold ratherthan Met¹⁷. CG-13 was relatively insensitive to N-terminal deletion ofNPY and PYY; the affinity for porcine NPY₂₂₋₃₆ was only 5-fold less thanthat for full length porcine NPY. Extreme deletion of α-helicalstructure was less well tolerated; the affinity for porcine NPY₂₆₋₃₆ was240-fold less than that for full length porcine NPY. Human [Leu³¹,Pro³⁴]NPY and human PP, both having Pro³⁴ rather than Glu³⁴, did not bindwell (K_(i) >300 nM). Hydrolysis of the carboxyl terminal amide to freecarboxylic acid, as in NPY free acid, also disrupted binding affinityfor CG-13 (K_(i) >300 nM). The terminal amide appears to be a commonstructural requirement for pancreatic polypeptide family/receptorinteractions.

The competitive displacement data indicate that CG-13 binds PYY withequal or greater affinity than NPY. The C-terminal region of NPY is theprimary pharmacophore. CG-13 does not tolerate exchange of Gln³⁴ withPro³⁴, as revealed by low affinity interactions with human [Leu³¹,Pro³⁴]NPY and human PP. The binding profile, which is shared by SK-N-Be(2)cell receptors but not by human Y1 receptors, is characteristic of thepharmacologically defined Y2 receptor (refs. 2, 8; see also Table 2).The membrane binding studies therefore confirm and extend our assessmentthat CG-13 encodes a human Y2 receptor.

The pharmacological profile of the human Y2 receptor was furtherinvestigated using peptide analogs related to NPY, PYY, and PP (Table4). CG-13 did not discriminate human and frog analogs of NPY (K_(i)=0.74 and 0.87 nM, respectively), human and porcine analogs of NPY₂₋₃₆(K_(i) =2.0 and 1.2 nM, respectively), human and porcine analogs of[Leu³¹,Pro³⁴ ]NPY (K_(i) >130 and>540 nM, respectively), or human NPYand human [Tyr-o-Me²¹ ]NPY (K_(i) =0.74 and 1.6 nM, respectively). Thislast derivative was tested based on the proposal that it was selectivefor central vs. peripheral NPY receptors, with high binding affinity inrat CNS but low potency in rat vas deferens relative to NPY (83). Forthe receptors under investigation, however, [Tyr-O-Me²¹ ]NPY and humanNPY yielded highly similar binding profiles. The NPY derivative withgreatest selectivity for CG-13 was C2-NPY, a C² to C²⁷disulfide-stabilized derivative of NPY with an 8-amino-octanoic linkerreplacing NPY₅₋₂₄ (K_(i) =3.5 nM, ≧20-fold selective for CG-13 over Y1and Y4 receptors). C2-NPY has been described as a Y2-selective compound(3).

Three additional PYY derivatives yielded distinctive binding profiles.CG-13 bound with highest affinity and greatest selectivity to humanPYY3-36 (K_(i) =0.70 nM, ≧20-fold selective for CG-13 over Y1 and Y4receptors). PYY₃₋₃₆ is a major form of PYY-like immunoreactivity inblood and could therefore mediate CG-13-dependent processes in vivo (84,85). Porcine PYY was relatively nonselective and similar in bindingaffinity to human PYY (K_(i) =0.35 nM and 0.36 nM, respectively). Human[Pro³⁴ ]PYY was lacking in binding affinity for CG-13 (K_(i) >310),further supporting the argument that Pro³⁴ is disruptive for highaffinity peptide binding to the CG-13 receptor.

Six additional PP derivatives were investigated. Those peptides whichresemble human PP in that they contain Pro³⁴ (bovine, rat, avian, andfrog PP) displayed no activity in the CG-13 binding assay. High affinitybinding was detected only for salmon PP (K_(i) =0.17 nM), which isdistinguished by containing Gln³⁴. When the C-terminus of human PP wasmodified to more closely resemble human NPY, as in [Ile³¹, Gln³⁴ ]PP,the binding affinity for CG-13 was increased dramatically (K_(i) =20nM). It has been reported previously that [Ile³¹, Gln³⁴ ]PP was moreactive than PP in Y2 binding assays, while exhibiting decreased potencyfor putative PP receptors in rat vas deferens (86).

Several proposed NPY antagonists were analyzed for their ability to bindto CG-13 receptors. These include PYX-1 and PYX-2, C-terminalderivatives of NPY reported to antagonize NPY-mediated feeding andneurotransmitter release (87, 88, 89). Neither synthetic peptide boundto CG-13 with high affinity or selectivity (K_(i) =684 for PYX-1 andK_(i) >1000 nM for PYX-2). [D-Trp³² ]NPY is an NPY derivative reportedto regulate feeding behavior when injected into the hypothalamus of rats(90); this analog was inactive in the CG-13 binding assay. Anotherinactive compound was NPY₁₋₂₄ amide, a peptide reported to antagonizeNPY in the rat vas deferens (83).

Human Tissue Y2 Receptor Macrolocalization: PCR

Human Y2 mRNA was detected by PCR techniques in a broad range of humantissues (Table 5). Relatively intense hybridization signals weredetected in total brain, thoracic artery, coronary artery, and penis,with more moderate levels in frontal brain, ventricle, mesentery,stomach and ileum. Relatively low levels were detected in nasal mucosaand pancreas. Several other tissues were negative for Y2 mRNA asmeasured by this technique, including atrium, liver, and uterus.

Cloning and Expression of Two Isoforms of the Rat NPY/PYY (Y2) Receptor

Two rat genomic clones homologous to the human Y2 receptor wereisolated, termed rs5a (FIG. 8) and rs26a (FIG. 9). The nucleotidesequence of rs5a is 86.5% identical in the coding region to that of thehuman Y2 receptor (FIG. 10), and can encode a 381 amino acid proteinwith 94.5% identity to the human Y2 amino acid sequence (FIG. 11). Inthe putative transmembrane domains (TMs), the protein predicted by rs5aexhibits 98.2% amino acid identity with the human Y2 receptor (FIG. 11).This high degree of primary sequence identity is often observed forspecies homologues, and strongly suggests that the receptor encoded byrs5a is the rat Y2 receptor. However, even a single amino acidsubstitution can influence the functional properties of a receptor;thus, even species homologues exhibiting a high level of sequenceidentity may display different pharmacological properties (infra),underscoring the importance of obtaining both rat and human receptorsfor use in drug development.

Sequence analysis of the second genomic clone revealed that rs26a alsoencoded a full-length rat Y2 receptor; however, rs26a contains twonucleotide changes when compared with the sequence of rs5a. Bothnucleotide changes result in amino acid substitutions in the predictedrat Y2 receptor protein. With two (2) amino acid changes, the proteinencoded by rs26a is 99.7% identical to that of rs5a. Compared with thehuman Y2 receptor, the nucleotide sequence identity of rs26a is 85.2%and the amino acid sequence identity is 98.2%. This clone thereforeencodes an isoform of the rat Y2 receptor distinct from that encoded byrs5a. The locations of the amino acid substitutions (N-terminus and 5/6loop; see FIG. 3) suggest that they could potentially influence receptorfunction. The Y2 receptors encoded by rs5a and rs26a are likely torepresent allelic variants at the same gene locus; however, rs26a couldrepresent a second rat Y2 gene. Accordingly, we have designated theisoform encoded by rs5a as the rat Y2a receptor, and designated theisoform encoded by rs26a as the rat Y2b receptor.

The primary sequences of rat and human Y2 receptors, while highlyrelated, show distinct patterns of sequence motifs for N-linkedglycosylation, N-myristoylation, and protein phosphorylation. Forexample, the rat Y2a differs from the rat Y2b in that it contains anadditional site for phosphorylation by protein kinase C. Further, thehuman Y2 differs from both rat Y2 isoforms in containing two additionalsites for N-linked glycosylation, two additional sites for cAMP- andcGMP-dependent protein phosphorylation, an additional site for caseinkinase II phosphorylation, one additional site for protein kinase Cphosphorylation, and two fewer sites for N-myristoylation. These sitescould mediate differences in the function or regulation of the threereceptors. The isolation of two rat homologues of the Y2 receptorprovides the means to compare the pharmacological properties of the ratand human Y2 receptors (see below) in relation to their observeddifferences in primary structures. These data will be critical to thedesign and testing of human therapeutic agents acting at these sites.

Binding Studies with Rat Y2 Homologs

The DNA corresponding to the rat Y2a homolog was transiently expressedin COS-7 cells for membrane binding studies. The binding of ¹²⁵ I-PYY tothe rat Y2a receptor was saturable over a radioligand concentration of0.5 pM to 2.5 nM. Binding data were fit to a one-site model with anapparent K_(d) =0.26 nM and a receptor density of 5100 fmol/mg membraneprotein. As determined by using peptide analogs within the pancreaticpolypeptide family, the rat Y2a pharmacological profile resembles thatfor the human Y2 receptor (Table 6). Each receptor analog is relativelytolerant of N-terminal ligand deletion (the human apparently more sothan the rat) and intolerant of any peptide containing Pro³⁴ or amodified C-terminus (as in NPY free acid or [D-Trp³² ]NPY).

The rat Y2b clone, which differs from rat Y2a by two amino acid changesone in the N-terminal tail (from Leu²⁰ to Phe²⁰) and another in thethird intracellular loop (from Thr²⁶⁶ to Met²⁶⁶), has been subjectedonly to a preliminary investigation. Membranes from COS-7 cellstransiently transfected with the rat Y2b receptor were incubated with0.08 nM ¹²⁵ I-PYY and analyzed for specific binding after incubation at30° C. for 120 min. Membranes from transfected cells bound 310 fmol ¹²⁵I-PYY/mg membrane protein, whereas membranes from mock-transfected cells(receiving vector without receptor cDNA insert) bound only 3 fmol ¹²⁵I-PYY/mg membrane protein. It remains to be determined whether thereexist any pharmacological or functional differences between the ratY2aand rat Y2b receptors.

Localization of NPY Y2 Messenger RNA in the Rat Central Nervous System

In control experiments, hybridization signals for rat NPY Y2 mRNA wereseen only with the antisense probes (probe sequences shown in Table 7),and only over cells which had been transfected with the rat Y2 DNA (FIG.18). The probes were designed to recognize both rat Y2a and rat Y2b.Neither mock transfected cells nor cells transfected with rat NPY Y1mRNA exhibited hybridization signals. On rat brain sections, nohybridization signals were obtained with the sense probes, only with theantisense probes.

The distribution of NPY Y2 mRNA observed in coronal sections through therostrocaudal extent of rat brain is shown in FIG. 12 and Table 8.Hybridization signals were seen over many areas of the rat brain (FIG.12), which, at the microscopic level, were confined to the cytoplasm ofneuronal profiles (data not shown). In the telencephalon, the mostintense hybridization signals were observed over the CA3 region of thehippocampus (FIGS. 12B-E) and over the anteroventral aspect of themedial nucleus of the amygdala (FIGS. 12C, D). Less intense signals werefound over the olfactory tubercle, the lateral septal nucleus (FIG.12A), and over the basomedial nucleus and posteromedial cortical nucleusof the amygdala (FIGS. 12D, E). Scattered neurons with hybridizationsignal were also seen in the central amygdaloid nucleus. In cortex,silver grains were seen over large neurons in the piriform region.

Among diencephalic structures, the arcuate nucleus of the hypothalamusexhibited the most intense hybridization signal for NPY Y2 mRNA (FIGS.12D, E). In this area, most of the neurons appeared to be labelled, andmany neurons were also labelled in the region of the tuber cinereumlateral to the arcuate nucleus. In addition, both the dorsomedial andventromedial hypothalamic nuclei contained appreciable hybridizationsignals over subpopulations of neurons (FIGS. 12C, D). In the dorsal andventral premammillary nuclei, hybridization signal was seen over manyneurons (FIG. 12E). In the thalamus, neurons in the centromedial nucleuswere labelled (FIGS. 12C, D), while a smaller, less intensely labelledgroup of cells was visible in the paraventricular nucleus (FIG. 12D).

In the mesencephalon, medulla, and pons, few structures were labelledwith the antisense oligonucleotide probe. Those exhibiting a moderatelevel of hybridization signal were the dorsal and caudal linear raphe(FIG. 12F), the pontine nucleus, and the posterior dorsal tegmentalnucleus (FIG. 12G). In the spinal cord, labelling was observed overscattered large neurons in lamina 9 (FIG. 12H). Silver grains were alsofound over a few large neurons in the dorsal root ganglion.

Receptor/G Protein Interactions: Effects of Guanine Nucleotides

For a given G protein-coupled receptor, a portion of the receptorpopulation in a membrane homogenate typically exists in the highaffinity ligand binding state as a receptor/G protein complex. Thebinding of GTP or a nonhydrolyzable analog to the G protein causes aconformational change in the receptor which favors a low affinityligand/binding state (110). We investigated whether the non-hydrolyzableGTP analog, Gpp(NH)p, would alter the binding of human NPY or ¹²⁵ I-PYYto Y2 receptors transiently expressed in COS-7 cells. The competitioncurve produced by human NPY was evaluated in the absence and presence of100 μM Gpp(NH)p. The human Y2 receptor was relatively insensitive to theGpp(NH)p compared to the rat Y2a receptor (FIG. 13). The IC₅₀ for humanNPY binding to the human Y2 receptor was increased from 2.2 nM to 3.3nM; specific binding of ¹²⁵ I-PYY was decreased by only 4% (n=5). TheIC₅₀ for human NPY binding to the rat Y2a receptor was altered verylittle (from 0.7 nM to 1.2 nM, n=2); specific binding of ¹²⁵ I-PYY,however, was decreased by 23% (n=2). A similar pattern of sensitivity toGpp(NH)p was reported for ¹²⁵ I-PYY binding to rat brain (91). Thedifference between the rat and human Y2 receptor clones could beexplained by several factors, including 1) the types of G proteinsavailable in COS-7 cells, 2) the level of receptor reserve in COS-7cells (note that human Y2 receptor density was greater than that of therat Y2a receptor), and 3) the efficiency of receptor/G protein coupling(92; 93).

Stable Expression Systems: Characterization in Binding Assays

Untransfected 293 and NIH-3T3 cells were pre-screened for specific ¹²⁵I-PYY binding and found to be negative (data not shown). Afterco-transfection with the human Y2 cDNA plus a G-418-resistant gene andselection with G-418, surviving colonies were screened for specificbinding of ¹²⁵ I-PYY. Two positive clones were identified and isolatedfor further study (293 clone #10 and NIH-3T3 clone #5). The binding of¹²⁵ I-PYY to membranes from the 293 stable clone was saturable over aradioligand concentration range of 0.5 pM to 2.5 nM. Binding data werefit to a one-site binding model with an apparent K_(d) of 3±1 pM and areceptor density of 880±50 fmol/mg membrane protein (mean±s.e.m., n=3).Membranes from stably transfected NIH-3T3 cells displayed similarbinding properties, with an apparent K_(d) of 8±2 pM and a receptordensity of 160±60 fmol/mg membrane protein (mean±s.e.m., n=2). Membranesfrom both stable clones were incubated with 0.08 nM ¹²⁵ I-PYY in thepresence or absence of 100 μM Gpp(NH)p. Specific binding of ¹²⁵ I-PYY toY2 receptors in 293 cell membranes was reduced 32% in the presence ofthe guanine nucleotide, whereas specific binding to Y2 receptors inNIH-3T3 cell membranes was reduced only 6% under the same conditions.The data serve to emphasize that the receptor/G protein interactions fora given receptor clone can vary depending upon the resident G proteinsin the host cell line (93). Additional factors such as receptor densityand receptor reserve can also play a role (92).

Functional Assay: cAMP

Activation of all Y-type receptors described thus far is thought toinvolve coupling to G-proteins which are inhibitory for adenylatecyclase activity (G_(i) or G_(o)) (1). Based on these priorobservations, we investigated the ability of PYY to inhibitforskolin-stimulated cAMP accumulation in 293 cells stably expressingthe human Y2 receptor. Incubation of intact cells with 10 μM forskolinproduced a 10-fold increase in cAMP accumulation over a 5 minute period,as determined by radioimmunoassay. Simultaneous incubation with humanPYY decreased the forskolin-stimulated cAMP accumulation by 71% instably transfected 293 cells (FIG. 14) but not in untransfected cells(data not shown). The NPY-mediated response was concentration-dependent(EC₅₀ =0.25 nM). We conclude that human Y2 receptor activation canresult in decreased cAMP accumulation, very likely through inhibition ofadenylate cyclase activity. Similar results were obtained for NIH-3T3cells stably transfected with the human Y2 receptor, in which human NPYdecreased forskolin-stimulated cAMP accumulation by 50% in transfectedcells with an EC₅₀ of 0.21 nM (FIG. 14).

Peptides selected for their ability to bind to the transiently expressedhuman Y2 receptor were further investigated for functional activityusing stably transfected 293 cells (Table 9). All peptides withmeasurable binding affinity were able to mimic the effects of PYY oncAMP accumulation. EC₅₀ values were generally within a 10-fold range ofK_(i) values, often lower in magnitude (Table 9). We also investigatedthe functional activity of the reported feeding behavior modulator[D-Trp³² ]NPY. Consistent with this peptide's low binding affinity forthe human Y2 receptor, we detected no functional activity atconcentrations up to 0.3 μM, or when tested at 0.3 μM for antagonism ofthe functional response (data not shown). The reported NPY receptorantagonists PYX-1 and PYX-2 were also inactive when tested under thesame paradigm.

Functional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was increased in 293 cellsstably transfected with the human Y2 receptor after application of 1 μMhuman PYY (Δ[Ca²⁺ ]_(i) =80 nM; FIG. 15). The PYY-mediated response wasconcentration-dependent, with EC₅₀ =39 nM, n=2 (FIG. 15). PYY-inducedcalcium mobilization was relatively maintained in the presence of 1 mMextracellular EGTA (Δ[Ca^(2+]) _(i) =64 nM for 1 μM human PYY),suggesting that intracellular calcium stores are the primary source ofthe transient calcium flux. Pretreatment with pertussis toxin (100 ng/mlfor 24 hours) decreased the response to 300 nM human PYY by 93%, therebysupporting a G protein-linked signal transduction pathway. Untransfected293 cells did not respond to human PYY (data not shown). The calciummobilization assay provides a second pathway through which Y2 receptoractivation can be measured.

DISCUSSION

Attempts to isolate the NPY Y2 receptor subtype based on sequencehomology with the Y1 receptor have not been successful so far.Therefore, we chose an expression cloning approach where a functionalreceptor is actually detected with exquisite sensitivity on the surfaceof transfected cells, using a highly specific iodinated ligand. Usingthis strategy, we have identified a human cDNA encoding thepharmacologically defined Y2 receptor. The fact that we had to screen2.2×10⁶ independent clones with a 3 kb average insert size to find oneclone reveals either a very strong bias against Y2 cDNA cloning in thecDNA library construction procedure, or the Y2 mRNA is expressed at verylow levels in human hippocampal tissue. The longest reading frame in thecDNA encodes a 381 amino acid protein with an estimated molecular weightof 42 kD. Given the fact that there is an N-linked glycosylation site inthe amino terminus, the apparent molecular weight could be slightlyhigher and in good agreement with published data on the molecular weightof the human hippocampal Y2 receptor at 50 kD (36). The Y2 receptorcarries a large number of potential phosphorylation sites which could beinvolved in the regulation of its functional characteristics. Thenucleotide and amino acid sequence analysis both reveal low identitylevels with all 7-TM receptors including the human Y1 and Y4 receptors.The highest transmembrane amino acid identity is found with the mouseMUSGIR receptor. A pharmacological profile on the human GIR homolog willbe established with NPY, PYY and pancreatic polypeptide related ligandsto find out if this orphan receptor belongs to the samepharmacologically defined neuropeptide Y receptor sub-family. The humanY2 receptor shares very low amino acid identity with the previouslycloned human Y1 receptor (31% overall and 41% in transmembrane regions).The human Y2 receptor also displays a unique pharmacological profile anda unique time course of association with ¹²⁵ I-PYY. The dramaticdifferences in sequence and pharmacological profile between the human Y1and Y2 receptors suggest that they might be encoded by two unrelatedgenes whose products have evolved into binding the same family ofligands. Conversely, they could have diverged from a common ancestorvery early in evolution and undergone multiple mutations leading todistinct pharmacological characteristics.

Northern analysis reveals a 4.3 kb band in human brain and demonstratesthat our 4.2 kb Y2 cDNA is full-length. Southern analyses are consistentwith the human genome containing a single Y2 receptor gene.

The pharmacological binding profile established in our initialcharacterization served primarily to establish the CG-13 as a human Y2receptor. The additional data included here reflect an increasedunderstanding of receptor ligand/interactions. We now know, for example,that C2-NPY and PYY₃₋₃₆ can be used to compete for Y2 receptor siteswith greater affinity and selectivity than the C-terminal fragments ofNPY originally described. We also know that certain peptides which arethought to antagonize NPY-dependent effects, such as [D-Trp³² ]NPY,PYX-1, and PYX-2, are unable to compete for binding of the human Y2receptor clone described here. Our evidence does not therefore supportthe cloned Y2 receptor as the molecular target of these particularpeptides in vivo or in vitro.

Human Y2 receptor mRNA was detected by PCR techniques in a broad rangeof human tissues (Table 5). Relatively intense hybridization signalswere detected in total brain, thoracic artery, coronary artery, andpenis, with more moderate levels in frontal brain, ventricle, andmesentery. This distribution is consistent with evidence for Y2 receptorlocalization and receptor-dependent effects in CNS, cardiovascular, andreproductive physiology (94). Moderate hybridization signals were alsodetected in stomach and ileum, consistent with evidence for Y2-mediatedeffects on chief cell cAMP accumulation (95) and also intestinalelectrolyte flux (61; 96). Relatively low levels were detected in nasalmucosa and pancreas, two tissues in which Y2-like receptors have beenreported to regulate vasoconstriction and pancreatic secretion,respectively (97, 98, 99). A more definitive localization of the Y2receptor mRNA and receptor expression (i.e., whether on neurons,enterocytes, vascular smooth muscle cells, etc.) is attainable throughin situ hybridization and receptor autoradiography techniques.

The distribution of NPY Y2 mRNA described here in rat brain has a numberof potential implications, and raises a number of important questions.Among these are; 1) how does the distribution of this mRNA correlatewith that of NPY itself; 2) how does the Y2 mRNA distribution relate tothe putative autoradiographic localization of Y2 receptors described byprevious investigators; and 3) what are the functional implications ofthe Y2 mRNA distribution?

Correlation with NPY Immunoreactivity

Neuropeptide Y is one of the most abundant and widely distributedpeptides in the mammalian brain (100). In some areas, NPY Y2 mRNAappears to be co-distributed with NPY-immunoreactive (NPYir) neurons,although colocalization in the same neuron(s) remains to be established.In both the arcuate nucleus of the hypothalamus and the medial nucleusof the amygdala, the distribution of Y2 mRNA overlaps with thedistribution of NPYir neurons demonstrated by immunocytochemical studies(100, 101). In addition, both areas contain moderate plexuses of NPYiraxons. These observations leave open the question ofpresynaptic/postsynaptic nature of the Y2 receptor. In most other areasof the brain, the Y2 mRNA does not appear to be co-distributed withNPYir neurons, but instead correlates better with the distribution ofNPYir terminal fields, suggesting a postsynaptic localization.

Comparison with Receptor Autoradiography

A number of investigators have described the distribution of NPYreceptors based on the autoradiographic localization of radiolabelledNPY ligands, among them [¹²⁵ I]NPY and [¹²⁵ I]peptide YY (PYY), incombination with subtype-selective displacers. The Y2 receptor has beenlocalized by combining [¹²⁵ I]PYY with the Y2-selective mask NPY₁₃₋₃₆(94). The results of such studies suggest that the Y2 receptor is widelydistributed in rat brain, being most abundant in the hippocampus,olfactory bulb, and hypothalamus. We have seen no NPY Y2 mRNA in theolfactory bulb, but both hippocampus and hypothalamus contain Y2 mRNA.However, the pharmacological characterization of NPY receptor subtypesis incomplete at present, and some of the Y2-like binding may beattributable to the so-called atypical Y1 receptor, or to otherundiscovered NPY receptor subtypes. Our in situ results suggest that thereceptor autoradiographic characterization of the Y2 receptor is likelyto be accurate for some areas. The projection fields of neuronscontaining the Y2 mRNA are important in this respect. Thus the pyramidalneurons of the CA3 region of the hippocampus, which contain relativelyintense Y2 hybridization signals, project in a topographic fashion tothe lateral septum (102), an area which supposedly contains a highproportion of Y2 receptors (103, 23, 94). Similarly, the olfactory bulbappears to contain mainly NPY receptors of the Y2 subtype. While thereis no Y2 mRNA in the olfactory bulb, the piriform cortex contains manyneurons which are labelled with the Y2 antisense probe, and provides amajor source of olfactory bulb afferents. The localization of NPY Y2mRNA in the arcuate nucleus of the hypothalamus is particularlyinteresting, as NPYir neurons in this nucleus provide the NPYinnervation of much of the hypothalamus, including the paraventricularand dorsomedial nuclei (104, 105). It is unclear at present whichreceptor subtype(s) predominate in the paraventricular nucleus, butbased on our results with the Y2 mRNA, and those of Mikkelsen andcolleagues with the Y1 mRNA (106, 107), both Y1 and Y2 should bepresent. Similar arguments can be pursued for most of the regions whichcontain Y2 mRNA, however a definitive profile of Y2 receptorlocalization awaits the introduction of Y2 selective ligands.

Functional Considerations

Neuropeptide Y is involved in a number of physiological functions,including the regulation of food intake, neuronal excitability,cardiovascular regulation, and circadian rhythms. With regard to foodintake, the paraventricular nucleus of the hypothalamus is one sitewhich has been intensively investigated, and has been demonstrated to bea prominent locus of action for the orexigenic effects of NPY. Thelocalization of NPY Y2 mRNA in the arcuate nucleus, and the projectionsof the arcuate to the paraventricular nucleus, suggest the involvementof this receptor in feeding.

In the hippocampus, NPY immunoreactivity is found mainly in interneuronswhich innervate pyramidal cells. Here, NPY has been demonstrated toreduce synaptic excitation in areas CA1 and CA3. This has been assumedto be mediated by a Y2 receptor (108), as C-terminal fragments of NPYare effective in the assay. The localization of Y2 mRNA in pyramidalcells of CA3 indicates that this receptor may be involved in thetermination of convulsive activity, such as in epilepsy.

The rat Y2a and Y2b receptor analogs represent essential tools forpharmaceutical drug development. Drug candidates screened primarilyagainst human receptors must also be characterized at the rat (or otherrelevant species analog) so that data generated from in vivo models canbe interpreted accurately. While the current panel of peptides revealedno major differences in pharmacological profile between the human Y2 andrat Y2a receptor analogs, even a single amino acid difference betweenreceptors displaying high sequence similarity could have dramaticeffects on ligand binding affinity (109). The rat Y2b receptorrepresents an additional opportunity to evaluate species-dependentdifferences in ligand binding. It remains to be determined whether therat Y2b receptor plays a singular role in rat Y2 receptor pharmacology,due either to unique ligand binding properties or to distinctivelocalization patterns.

We established functional assays for human Y2 receptor activation inboth 293 and NIH-3T3 cells based on receptor-dependent inhibition offorskolin-stimulated cAMP accumulation (Table 9). The EC₅₀ values forpeptides in these assays were generally smaller than the correspondingK_(i) values, suggesting that receptor activation occurs through a highaffinity state of the receptor which is not predominantly representedunder the conditions of the binding assay. Such a scenario would beconsistent with the weak effect of Gpp(NH)p on radioligand binding tothe human Y2 receptor in membrane homogenates.

Our characterization of the Y2 receptor stably expressed in 293 cellsalso shows definitively that the Y2 receptor can couple simultaneouslyto both cAMP regulation and calcium mobilization in a single cell type.The calcium mobilization in 293 cells, at least, appears to occurthrough a pertussis toxin-sensitive G protein. The EC₅₀ for the humanPYY-mediated calcium response is significantly larger than that for thecAMP response in the same host cell (39 nM vs. 0.31 nM, respectively),suggesting that calcium mobilization requires promiscuous coupling ofthe receptor to a G protein other than that involved in cyclaseregulation. The exact identities of the G proteins mediating thesereceptor activation events, whether G_(i), G_(o), G_(z) or another type,remain to be determined.

We now have several Y2 receptor expression systems from which to choose,each uniquely suited to different uses. The transient expression systemin COS-7, for example, allows us to generate sufficient quantities ofmembranes for routine structure/activity relationship measurements. Wecan also produce mutant receptors by site-directed mutagenesis orrelated enzymatic techniques and express them transiently in COS-7 for acomparison of pharmacological properties with those of the wild-typereceptor. In this way, we can gain insight into receptor bindingpockets, ligand binding domains, and mechanisms of activation. Thestable expression system in 293 and NIH-3T3 cells offers the convenienceof a single transfection followed by routine passaging techniques. Thestable expression system also offers the opportunity to select foroptimum receptor expression levels, G protein populations, and signaltransduction pathways, all of which are critical elements for in vitrofunctional assays. Such assays can be used to determine agonist orantagonist activity in receptor-selective compounds, thereby generatingcritical information for drug design.

The expression cloning of a human Y2 receptor allows, for the firsttime, the ability to develop NPY-receptor subtype specific drugs andrepresents a major advance in our ability to analyze NPY-mediatedphysiological processes. Pharmacologically defined Y2 receptors have awidespread anatomical distribution (2). They represent the predominantNPY receptor in brain, with the highest density in hippocampus andrelatively high expression in almost all other areas including olfactorybulb, basal ganglia, amygdaloid complex, thalamic and hypothalamicnuclei, pituitary, pineal gland, cerebellum, and brainstem. Thisdistribution is consistent with northern blot analysis, which shows thatthe Y2 MRNA is present in amygdala, candate nucleus, corpus callosum,hippocampus, hypothalamus, substantia nigra and subthalamic nucleus.Peripheral localization includes sympathetic neurons, dorsal rootganglia, stomach chief cells, intestinal enterocytes, kidney proximaltubule, trachea, and vascular smooth muscle. Y2 receptors are thereforein a position to potentially regulate a variety of physiologicalfunctions including cognitive enhancement, circadian rhythm, EEGsynchronization, body temperature, blood pressure, locomotor activity,neuroendocrine release, sympathetic activation, sensory transmission,gastrointestinal function, intestinal secretion, renal absorption, andcardiovascular function (1, 2).

Y2 receptors are attractive targets for drug design (1). Y2 receptorregulation may be useful in the treatment of several pathophysiologicalconditions (1, 2) including memory loss (111), epileptic seizure (72),pain (64), depression, hypertension, locomotor problems, sleepdisturbances, eating disorders, sexual/reproductive disorders, nasalcongestion (97), and diarrhea (112). A rigorous investigation ofY2-related pathophysiology has been hindered by the absence of suitablenon-peptide ligands. The chemical synthesis of subtype selectiveagonists and antagonists as potential drug candidates will be greatlyaccelerated by screening against a homogeneous population of clonedhuman Y2 receptors. As more specific pharmacological tools becomeavailable for probing receptor function, additional therapeuticindications are likely to be discovered.

We do not know whether the human and rat Y2 receptors we have discoveredaccount for all of the pharmacological Y2 receptors so far described, orwhether the Y2 receptor population is further divided into distinctreceptor subtypes. Indeed, there is some suggestion of receptorheterogeneity within the Y2 receptor population (2). These are issueswhich can now be resolved using nucleotide sequence from the human Y2receptor as the basis for in situ localization, anti-sense strategies,homology cloning, and related techniques. Such approaches will enable usto investigate the existence of potentially novel NPY receptor subtypes,in humans and other species, with additional pharmacologic andtherapeutic significance.

                  TABLE 1                                                         ______________________________________                                        % aminoacid TM identity of the NPY-2 receptor                                 with other 7 TM Receptors                                                     ______________________________________                                        m MUSGIR     42      h Y-1        41                                                               h Y-4        41                                          h 5HT1A      28      h Adenosine A2b                                                                            28  h                                       Substance K  33                                                               h 5HT2       31      h Adenosine A1                                                                             29  h                                       Substance p  32                                                               h α-adrenergic-1b                                                                    34      h Dopamine D1                                                                              31  h                                       Neurokinin-3 33                                                               h α-adrenergic-2a                                                                    34      h Dopamine D2                                                                              32  h                                       Interleukin-8                                                                              33                                                               h β-adrenergic-1                                                                      35      bov Hist H1  25  h                                       Angiotensin.sub.1                                                                          33                                                                                    h Hist H2    28  h                                       Angiotensin.sub.2                                                                          27                                                                                                     m                                       Thyrotropin  27                                                               releasing hormone                                                                                                   h                                       Bradykinin   25                                                                                                     r                                       mas oncogene 20                                                               ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Pharmacologically defined receptors for NPY and related pancreatic            polypeptides.                                                                 Rank orders of affinity are based on published reports of binding and         functional data                                                               (M9, M24, M3, M10). Missing peptides in the series reflect a lack of          published information.                                                        Affinity (-pk.sub.i or -pEC.sub.50)                                           Receptor                                                                           11 to 10 10 to 9                                                                            9 to 8   8 to 7                                                                             7 to 6                                                                           <6                                        __________________________________________________________________________    Y1   NPY           NPY.sub.2-36                                                                           NPY.sub.13-36                                                                      PP                                                PYY                                                                           [Leu.sup.31,Pro.sup.34 ]NPY                                              Y2            PYY  NPY.sub.13-36    [Leu.sup.31,Pro.sup.34 ]NPY                             NPY                   PP                                                      NPY.sub.2-36                                                    Y3            NPY  [Pro.sup.34 ]NPY                                                                       NPY.sub.13-36                                                                         PYY                                                                   PP                                                PP   PP            [Leu.sup.31,Pro.sup.34 ]NPY                                                                    NPY                                       __________________________________________________________________________

                  TABLE 3                                                         ______________________________________                                        Pharmacological profile of the CG-13 receptor.                                Binding data reflect competitive displacement of .sup.125 I-PYY from          membranes of COS-7 cells transiently expressing CG-13 receptors.              Peptides were tested at concentrations ranging from 0.001 nM to               100 nM. IC.sub.50 values corresponding to 50% displacement were               determined by nonlinear regression analysis and converted to K.sub.i          values                                                                        according to the equation, K.sub.i = IC.sub.50 /(1 + [L]/K.sub.d), where      [L] is                                                                        the .sup.125 I-PYY concentration and K.sub.d is the equilibrium               dissociation                                                                  constant of .sup.125 I-PYY. The data shown are representative of at           least                                                                         two independent experiments                                                                                    SK-N-                                                 Human Y1,     CG-13,    Be(2),                                       Competitor                                                                             K.sub.i (nM)  K.sub.i (nM)                                                                            K.sub.i (nM)                                 ______________________________________                                        human PYY                                                                               0.085 ± 0.021                                                                           0.39 ± 0.05                                                                          0.11 ± 0.02                               human NPY                                                                               0.049 ± 0.009                                                                           0.69 ± 0.14                                                                          0.13 ± 0.02                               porcine   1.4 ± 0.2 0.78 ± 0.13                                                                          0.41 ± 0.09                               NPY.sub.2-36                                                                  porcine NPY                                                                             0.049 ± 0.001                                                                           0.86 ± 0.13                                                                          0.28 ± 0.04                               porcine  32 ± 7     1.5 ± 0.2                                                                            0.86 ± 0.14                               PYY.sub.13-36                                                                 porcine  28 ± 5     1.5 ± 0.2                                                                            2.1 ± 0.5                                 NPY.sub.18-36                                                                 porcine   51 ± 16   2.4 ± 0.4                                                                            1.8 ± 0.4                                 NPY.sub.13-36                                                                 porcine  62 ± 6     3.4 ± 0.3                                                                            3.1 ± 0.6                                 NPY.sub.20-36                                                                 porcine  45 ± 4     3.8 ± 0.7                                                                            5.0 ± 0.5                                 NPY.sub.16-36                                                                 porcine  170 ± 30   4.6 ± 0.1                                                                            3.2 ± 0.6                                 NPY.sub.22-36                                                                 porcine  >300          210 ± 60                                                                             70 ± 7                                    NPY.sub.26-36                                                                 human NPY                                                                              >300          >300      280 ± 120                                 free acid                                                                     human PP 200 ± 70   >300      >300                                         human     0.13 ± 0.02                                                                             >300      >300                                         [Leu.sup.31,Pro.sup.34 ]                                                      NPY                                                                           ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Extended pharmacological binding profile of                                   the human Y2 receptor vs. other Y-type receptors cloned from human.           Binding data reflect competitive displacement of .sup.125 I-PYY from          membranes of COS-7 cells transiently expressing human Y1, human               Y2, and human Y4 receptors. IC.sub.50 values corresponding to 50%             displacement were determined by nonlinear regression analysis and             converted to K.sub.i values according to the equation Chang-Prusoff           equation, K.sub.i = IC.sub.50 /(1 + [L]/K.sub.d), where [L] is the            .sup.125 I-PYY                                                                concentration and K.sub.d is the equilibrium dissociation constant of         .sup.125 I-                                                                   PYY. Any peptide not included in initial characterization shown in            previous tables is referred to as a "new peptide". Data shown are             representative of at least two independent experiments.                       Peptide   Y1        Y2        Y4      Comments                                ______________________________________                                        NPY, human                                                                              0.08      0.74      2.2                                             NPY,      0.07      0.81      1.1                                             porcine                                                                       NPY, frog 0.07      0.87      1.2     new                                     (melanostatin)                        peptide                                 O--Me-Tyr.sup.21 -                                                                      0.12      1.6       6.1     new                                     NPY, human                            peptide                                 C2-NPY,   73        3.5       120     new                                     porcine                               peptide                                 NPY.sub.2-36,                                                                           3.6       2.0       16      new                                     human                                 peptide                                 NPY.sub.2-36,                                                                           2.4       1.2       5.6                                             porcine                                                                       NPY.sub.13-36,                                                                          70        2.5       38                                              porcine                                                                       NPY.sub.16-36,                                                                          41        3.6       54                                              porcine                                                                       NPY.sub.18-36,                                                                          70        4.2       >300                                            porcine                                                                       NPY.sub.20-36,                                                                          63        3.6       120                                             porcine                                                                       NPY.sub.22-36,                                                                          >1000     18        >990                                            porcine                                                                       NPY.sub.26-36,                                                                          >1000     380       300                                             porcine                                                                       [Leu.sup.31,                                                                            0.15      >130      1.1                                             Pro.sup.34 ]NPY,                                                              human                                                                         [Leu.sup.31,                                                                            0.15      >540      1.5     new                                     Pro.sup.34 ]NPY,                      peptide                                 porcine                                                                       NPY free  490       >1000     >1000                                           acid, human                                                                   NPY.sub.1-24                                                                            >1000     >1000     >1000   new                                     amide,                                peptide                                 human                                                                         [D-Trp.sup.32 ]NPY,                                                                     >1000     >1000     >1000   new                                     human                                 peptide                                 PYY, human                                                                              0.19      0.36      0.87                                            PYY,      0.14      0.35      1.3     new                                     porcine                               peptide                                 PYY.sub.3-36,                                                                           45        0.70      14      new                                     human                                 peptide                                 PYY.sub.13-36,                                                                          33        1.5       46                                              porcine                                                                       [Pro.sup.34 ]PYY,                                                                       0.14      >310      0.12    new                                     human                                 peptide                                 PP, human 77        >1000     0.06                                            PP, bovine                                                                              240       >830      0.05    new                                                                           peptide                                 PP, rat   460       >1000     0.18    new                                                                           peptide                                 PP, avian 400       >1000     7.0     new                                                                           peptide                                 PP, frog  98        >1000     61      new                                                                           peptide                                 PP, salmon                                                                              0.20      0.17      3.2     new                                                                           peptide                                 [Ile.sup.31,                                                                            >86       20        0.09    new                                     Gln.sup.34 ]PP,                       peptide                                 human                                                                         PYX-1     507       684       794     new                                                                           peptide                                 PYX-2     >1000     >1000     >1000   new                                                                           peptide                                 ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Macrolocalization of human Y2 receptor mRNA in                                human tissues by PCR.                                                         Localization data reflect PCR-based amplification of human Y2 cDNA            derived from mRNA extracts of human tissues. Southern blots of the            PCR products were prepared and hybridized with .sup.32 P-labeled              oligonucleotide probes selective for Y-type receptor subtypes. The            labeled products were recorded on X-ray film and the relative signal          density was determined by visual inspection. In this rating scheme,           + = faint signal, ++ = moderate signal, +++ = intense signal.                 Human tissues  Human Y2 PCR Product                                           ______________________________________                                        total brain    +++                                                            frontal brain  ++                                                             ventricle (heart)                                                                            ++                                                             atrium (heart) (-)                                                            thoracic aorta +++                                                            coronary artery                                                                              ++1/2                                                          nasal mucosa   +                                                              mesentery      ++                                                             stomach        ++                                                             ileum          ++                                                             pancreas       +                                                              liver          (-)                                                            kidney         +                                                              bladder        +1/2                                                           penis          +++                                                            testes         not determined                                                 uterus         (-)                                                            (endometrium)                                                                 uterus (myometrium)                                                                          (-)                                                            ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Peptide binding profile of the rat Y2a                                        receptor vs. the human Y2 receptor.                                           Binding data reflect competitive displacement of .sup.125 I-PYY from          membranes of COS-7 cells transiently expressing rat Y2a and human             Y2 receptors. IC.sub.50 values corresponding to 50% displacement were         determined by nonlinear regression analysis and converted to K.sub.i          values                                                                        according to the equation Chang-Prusoff equation,                             K.sub.i = IC.sub.50 /(1 + [L]/K.sub.d), where [L] is the .sup.125 I-PYY       concentration and                                                             K.sub.d is the equilibrium dissociation constant of .sup.125 I-PYY. Data      shown are representative of at least two independent experiments.             Peptide          Rat Y2a  Human Y2                                            ______________________________________                                        NPY, human       1.3      0.74                                                NPY.sub.2-36, human                                                                            2.2      1.2                                                 NPY.sub.13-36, human                                                                           31       2.5                                                 NPY.sub.20-36, porcine                                                                         93       3.6                                                 NPY.sub.26-36, porcine                                                                         >830     380                                                 NPY free acid,   >980     >1000                                               human                                                                         [Leu.sup.31,Pro.sup.34 ]NPY,                                                                   >1000    >130                                                human                                                                         [D-Trp.sup.32 ]NPY,                                                                            >830     >1000                                               human                                                                         PYY, porcine     0.28     0.35                                                PYY.sub.13-36, porcine                                                                         1.5      28                                                  PP, human        >1000    >1000                                               PP.sub.31-36, human                                                                            >10000   >10000                                              PP, salmon       0.17     0.17                                                PP, bovine       >1000    >825                                                PP, rat          >1000    >1000                                               ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Oligonucleotide probe sequences used for in                                   situ hybridization                                                            Probe  Sequence          Location  Orientation                                ______________________________________                                        KS972  5'-GGC CCA TTA GGT GCA                                                                          NH.sub.2 -                                                                              sense                                             GAG GCA GAT GAG AAT                                                                             terminus                                                    CAA ACT GTA GAA GTG-                                                          3' SEQ ID NO:24                                                        KS974  5'-CAC TTC TAC AGT TTG                                                                          NH.sub.2 -                                                                              antisense                                         ATT CTC ATC TGC CTC                                                                             terminus                                                    TGC ACC TAA TGG GCC-                                                          3' SEQ ID NO:25                                                        KS973  5'-CGG AGG TGT CCA TGA                                                                          COOH      sense                                             CCT TCA AGG CTA AAA                                                                             terminus                                                    AGA ACC TGG AAG TCA-                                                          3' SEQ ID NO:26                                                        KS975  5'-TGA CTT CCA GGT TCT                                                                          COOH      antisense                                         TTT TAG CCT TGA AGG                                                                             terminus                                                    TCA TGG ACA CCT CCG-                                                          3' SEQ ID NO:27                                                        ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Distribution of NPY Y2 mRNA in the rat CNS.                                   Positive hybridization signals are indicated by "+"                           signs, no signal by "-", and a low signal by "+/-".                           Region               Hybridization                                            ______________________________________                                        Cortex                                                                        layer 2              -                                                        layer 6              -                                                        piriform             +                                                        entorhinal           -                                                        cingulate            -                                                        Olfactory bulb       -                                                        Anterior olfactory n.                                                                              -                                                        Basal ganglia                                                                 caudate-putamen      +/-                                                      n. accumbens         -                                                        olfactory tubercle   +                                                        globus pallidus      -                                                        islands of Calleja   -                                                        Septal area                                                                   lateral septum       +                                                        medial septum        -                                                        septohippocampal     -                                                        diagonal band n.     -                                                        Claustrum            -                                                        Dorsal endopiriform  -                                                        Hypothalamus                                                                  anterior             -                                                        paraventricular      +                                                        dorsomedial          +                                                        ventromedial         +                                                        arcuate              +                                                        lateral              -                                                        mammillary           +                                                        tuberal              +                                                        Thalamus                                                                      anterior nuclei      -                                                        paraventricular n.   +                                                        rhomboid n.          -                                                        reuniens n.          -                                                        mediodorsal n.       -                                                        ventral nuclei       -                                                        reticular n.         -                                                        centrolateral n.     -                                                        centromedial n.      +                                                        zona incerta         -                                                        lateral posterior n. -                                                        lateral dorsal n.    -                                                        posterior n.         -                                                        medial geniculate n. -                                                        dorsal lateral gen.  -                                                        ventral lateral gen. -                                                        habenula             -                                                        Hippocampus                                                                   CA1                  -                                                        CA2                  -                                                        CA3                  +                                                        subiculum            -                                                        presubiculum         -                                                        parasubiculum        -                                                        Dentate gyrus                                                                 granule cell layer   -                                                        polymorph layer      -                                                        Amygdala                                                                      anterior             -                                                        medial               +                                                        cortical             +                                                        amygdalohipp.        -                                                        basomedial           +                                                        basolateral          -                                                        lateral              -                                                        central              +                                                        bed nucleus          -                                                        Midbrain                                                                      superior colliculus  -                                                        inferior colliculus  -                                                        mes. trigeminal      -                                                        dorsal raphe         +                                                        caudal linear raphe  +                                                        median raphe         -                                                        raphe magnu          -                                                        substantia nigra     -                                                        central gray         -                                                        Pons/medulla                                                                  locus coeruleus      -                                                        subcoeruleus         -                                                        parabrachial n.      -                                                        facial n.            -                                                        pontine n.           +                                                        pontine ret. n.      -                                                        reticulotegmental    +                                                        A5                   -                                                        A7                   -                                                        gigantocellular      -                                                        lateral reticular n. -                                                        motor trigeminal     NA                                                       spinal trigeminal    NA                                                       medial vestibular    -                                                        solitarius           NA                                                       dorsal vagus         NA                                                       hypoglossal          NA                                                       Cerebellum                                                                    granule cell layer   -                                                        molecular layer      -                                                        Purkinje cells       -                                                        deep nuclei          -                                                        Spinal cord                                                                   dorsal horn          -                                                        ventral horn         +                                                        intermediolateral    -                                                        Dorsal root ganglia  +                                                        ______________________________________                                         Abbreviations                                                                 1-9 spinal cord laminae                                                       Arc arcuate n. hypothalamus                                                   BMP posterior basomedial n. amygdala                                          CA3 field CA3 of the hippocampus                                              CC central canal                                                              Cli caudal linear raphe n.                                                    CM centromedial n. thalamus                                                   DMH dorsomedial n. hypothalamus                                               DR dorsal raphe n.                                                            LSV lateral septum, ventral                                                   Me medial n. amygdala                                                         MeAV medial n. amygdala, anteroventral division                               PDTg posterior dorsal tegmental n.                                            Pir piriform cortex                                                           PMD dorsal premammillary n.                                                   PMCo posterior medial cortical n. amygdala                                    Pn pontine n.                                                                 PVH paraventricular n. hypothalamus                                           PVT paraventricular n. thalamus                                               TC tuber cinereum                                                             TuO olfactory tubercle                                                        VMH ventromedial n. hypothalamus                                         

                  TABLE 9                                                         ______________________________________                                        Functional activation of the human Y2 receptor                                and inhibition of cAMP accumulation.                                          K.sub.i values were derived from binding assays as described in Table 1.      Peptides were evaluated for binding affinity and then analyzed for            functional activity. Functional data were derived from radio-                 immunoassay of cAMP accumulation in stably transfected 293 cells              stimulated with 10 μM forskolin. The maximum inhibition of cAMP            accumulation relative to that produced by human NPY (E.sub.max) and           the concentration producing a half-maximal effect (EC.sub.50) were            determined by nonlinear regression. Data shown                                are representative of at least two independent experiments.                                    Function                                                                 Binding    EC.sub.50                                              Peptide     K.sub.i (nM)                                                                             (nM)     E.sub.max                                     ______________________________________                                        NPY, human  0.74       0.25     100%                                          NPY, porcine                                                                              0.81       0.20     113%                                          C2-NPY,     3.5        0.14     116%                                          porcine                                                                       NPY.sub.2-36, human                                                                       2.0        0.35      94%                                          NPY.sub.2-36,                                                                             1.2        1.2       96%                                          porcine                                                                       NPY.sub.13-36,                                                                            2.5        1.7      110%                                          porcine                                                                       NPY.sub.16-36,                                                                            3.6        1.8       92%                                          porcine                                                                       NPY.sub.18-36,                                                                            4.2        2.1       92%                                          porcine                                                                       NPY.sub.20-36,                                                                            3.6        3.2       77%                                          porcine                                                                       NPY.sub.22-36,                                                                            18         2.3       88%                                          porcine                                                                       [Leu.sup.31,                                                                              >130       >3000    not                                           Pro.sup.34 ]NPY,                determined                                    human                                                                         [Leu.sup.31,                                                                              >540       >3000    not                                           Pro.sup.34 ]NPY,                determined                                    porcine                                                                       [D-Trp.sup.32 ]NPY,                                                                       >1000      >3000    not                                           human                           determined                                    PYY, human  0.36       0.31     100%                                          PYY, porcine                                                                              0.35       0.16     103%                                          PYY.sub.3-36, human                                                                       0.70       0.22      99%                                          PYY.sub.13-36,                                                                            1.5        0.13     102%                                          porcine                                                                       [Pro.sup.34 ]PYY,                                                                         >310       >120     not                                           human                           determined                                    PP, salmon  0.17       0.07      79%                                          PYX-1       684        >3000    not                                                                           determined                                    PYX-2       >1000      >3000    not                                                                           determined                                    ______________________________________                                    

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103. Lynch, D. R., M. W. Walker, R. J. Miller, and S. H. Snyder (1989)Neuropeptide Y receptor binding sites in rat brain: Differentialautoradiographic localizations with ¹²⁵ I-peptide YY and ¹²⁵I-neuropeptide Y imply receptor heterogeneity. J. Neurosci. 9:2607-2619.

104. Bai, F. L., M. Yamano, Y. Shiotani, P. C. Emson, A. D. Smith, J. F.Powell, and M. Tohyama (1985) An arcuato-paraventricular and-dorsomedial hypothalamic neuropeptide Y-containing system with lacksnoradrenaline in the rat. Brain Res. 331:172-175.

105. Gustafson, E. L. and R. Y. Moore (1987) Noradrenaline andneuropeptide Y innervation of the rat hypothalamus are differentiallyaffected by 6-hydroxydopamine. Neurosci.Lett. 83:53-58.

106. Mikkelsen, J. D. and P. J. Larsen (1992) A high concentration ofNPY (Y1) -receptor mRNA-expressing cells in the rat arcuate nucleus.Neurosci.Lett. 148:195-198.

107. Larsen. P. J., S. P. Sheikh, C. R. Jakobsen, T. W. Schwartz, and J.D. Mikkelsen (1993) Regional distribution of putative NPY Y₁ receptorsand neurons expressing Y₁ mRNA in forebrain areas of the rat centralnervous system. Eur.J. Neurosci. 5:1622-1637.

108. Colmers, W. F. and D. Bleakman (1994) Effects of neuropeptide Y onthe electrical properties of neurons. Trends Neurosci. 17:373-379.

109. Hall, J. M., Caulfield, M. P., Watson, S. P., and Guard, S. (1993)Receptor subtypes or species homologues: relevance to drug discovery,Trends Pharmacol. 14: 376-383.

110. Birnbaumer, L. (1990) Transduction of receptor signal intomodulation of effector activity by G proteins: the first 20 years or so. . . FASEB J. 4: 3178-3188.

111. Morley, J. E. and Flood, J. F. (1991). Neuropeptide Y and memoryprocessing. Ann. N.Y. Acad. Sci. 611: 226-231.

112. Cox, H. and Cuthbert, A. W. (1990) The effects of neuropeptide Yand its fragments upon basal and electrically stimulated ion secretionin rat jejunum mucosa. Br. J. Pharmac. 101: 247-252.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 27                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1280 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 43..1185                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GACTCTTGTG CTGGTTGCAG GCCAAGTGGA CCTGTACTGA AA ATG GGT - # CCA ATA            54                                                                          #           Met Gly Pro Ile                                                   #             1                                                               - GGT GCA GAG GCT GAT GAG AAC CAG ACA GTG GA - #A GAA ATG AAG GTG GAA          102                                                                          Gly Ala Glu Ala Asp Glu Asn Gln Thr Val Gl - #u Glu Met Lys Val Glu           #  20                                                                         - CAA TAC GGG CCA CAA ACA ACT CCT AGA GGT GA - #A CTG GTC CCT GAC CCT          150                                                                          Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Gl - #u Leu Val Pro Asp Pro           #                 35                                                          - GAG CCA GAG CTT ATA GAT AGT ACC AAG CTG AT - #T GAG GTA CAA GTT GTT          198                                                                          Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Il - #e Glu Val Gln Val Val           #             50                                                              - CTC ATA TTG GCC TAC TGC TCC ATC ATC TTG CT - #T GGG GTA ATT GGC AAC          246                                                                          Leu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Le - #u Gly Val Ile Gly Asn           #         65                                                                  - TCC TTG GTG ATC CAT GTG GTG ATC AAA TTC AA - #G AGC ATG CGC ACA GTA          294                                                                          Ser Leu Val Ile His Val Val Ile Lys Phe Ly - #s Ser Met Arg Thr Val           #     80                                                                      - ACC AAC TTT TTC ATT GCC AAT CTG GCT GTG GC - #A GAT CTT TTG GTG AAC          342                                                                          Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Al - #a Asp Leu Leu Val Asn           #100                                                                          - ACT CTG TGT CTA CCG TTC ACT CTT ACC TAT AC - #C TTA ATG GGG GAG TGG          390                                                                          Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Th - #r Leu Met Gly Glu Trp           #               115                                                           - AAA ATG GGT CCT GTC CTG TGC CAC CTG GTG CC - #C TAT GCC CAG GGC CTG          438                                                                          Lys Met Gly Pro Val Leu Cys His Leu Val Pr - #o Tyr Ala Gln Gly Leu           #           130                                                               - GCA GTA CAA GTA TCC ACA ATC ACC TTG ACA GT - #A ATT GCC CTG GAC CGG          486                                                                          Ala Val Gln Val Ser Thr Ile Thr Leu Thr Va - #l Ile Ala Leu Asp Arg           #       145                                                                   - CAC AGG TGC ATC GTC TAC CAC CTA GAG AGC AA - #G ATC TCC AAG CGA ATC          534                                                                          His Arg Cys Ile Val Tyr His Leu Glu Ser Ly - #s Ile Ser Lys Arg Ile           #   160                                                                       - AGC TTC CTG ATT ATT GGC TTG GCC TGG GGC AT - #C AGT GCC CTG CTG GCA          582                                                                          Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly Il - #e Ser Ala Leu Leu Ala           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - AGT CCC CTG GCC ATC TTC CGG GAG TAT TCG CT - #G ATT GAG ATC ATC CCG          630                                                                          Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser Le - #u Ile Glu Ile Ile Pro           #               195                                                           - GAC TTT GAG ATT GTG GCC TGT ACT GAA AAG TG - #G CCT GGC GAG GAG AAG          678                                                                          Asp Phe Glu Ile Val Ala Cys Thr Glu Lys Tr - #p Pro Gly Glu Glu Lys           #           210                                                               - AGC ATC TAT GGC ACT GTC TAT AGT CTT TCT TC - #C TTG TTG ATC TTG TAT          726                                                                          Ser Ile Tyr Gly Thr Val Tyr Ser Leu Ser Se - #r Leu Leu Ile Leu Tyr           #       225                                                                   - GTT TTG CCT CTG GGC ATT ATA TCA TTT TCC TA - #C ACT CGC ATT TGG AGT          774                                                                          Val Leu Pro Leu Gly Ile Ile Ser Phe Ser Ty - #r Thr Arg Ile Trp Ser           #   240                                                                       - AAA TTG AAG AAC CAT GTC AGT CCT GGA GCT GC - #A AAT GAC CAC TAC CAT          822                                                                          Lys Leu Lys Asn His Val Ser Pro Gly Ala Al - #a Asn Asp His Tyr His           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - CAG CGA AGG CAA AAA ACC ACC AAA ATG CTG GT - #G TGT GTG GTG GTG GTG          870                                                                          Gln Arg Arg Gln Lys Thr Thr Lys Met Leu Va - #l Cys Val Val Val Val           #               275                                                           - TTT GCG GTC AGC TGG CTG CCT CTC CAT GCC TT - #C CAG CTT GCC GTT GAC          918                                                                          Phe Ala Val Ser Trp Leu Pro Leu His Ala Ph - #e Gln Leu Ala Val Asp           #           290                                                               - ATT GAC AGC CAG GTC CTG GAC CTG AAG GAG TA - #C AAA CTC ATC TTC ACA          966                                                                          Ile Asp Ser Gln Val Leu Asp Leu Lys Glu Ty - #r Lys Leu Ile Phe Thr           #       305                                                                   - GTG TTC CAC ATC ATC GCC ATG TGC TCC ACT TT - #T GCC AAT CCC CTT CTC         1014                                                                          Val Phe His Ile Ile Ala Met Cys Ser Thr Ph - #e Ala Asn Pro Leu Leu           #   320                                                                       - TAT GGC TGG ATG AAC AGC AAC TAC AGA AAG GC - #T TTC CTC TCG GCC TTC         1062                                                                          Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Al - #a Phe Leu Ser Ala Phe           325                 3 - #30                 3 - #35                 3 -       #40                                                                           - CGC TGT GAG CAG CGG TTG GAT GCC ATT CAC TC - #T GAG GTG TCC GTG ACA         1110                                                                          Arg Cys Glu Gln Arg Leu Asp Ala Ile His Se - #r Glu Val Ser Val Thr           #               355                                                           - TTC AAG GCT AAA AAG AAC CTG GAG GTC AGA AA - #G AAC AGT GGC CCC AAT         1158                                                                          Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Ly - #s Asn Ser Gly Pro Asn           #           370                                                               - GAC TCT TTC ACA GAG GCT ACC AAT GTC TAAGGAAGC - #T GTGGTGTGAA               1205                                                                          Asp Ser Phe Thr Glu Ala Thr Asn Val                                           #       380                                                                   - AATGTATGGA TGAATTCTGA CCAGAGCTAT GAATCTGGTT GATGGCGGCT CA - #CAAGTGAA       1265                                                                          #  1280                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 381 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Gly Pro Ile Gly Ala Glu Ala Asp Glu As - #n Gln Thr Val Glu Glu         #15                                                                           - Met Lys Val Glu Gln Tyr Gly Pro Gln Thr Th - #r Pro Arg Gly Glu Leu         #             30                                                              - Val Pro Asp Pro Glu Pro Glu Leu Ile Asp Se - #r Thr Lys Leu Ile Glu         #         45                                                                  - Val Gln Val Val Leu Ile Leu Ala Tyr Cys Se - #r Ile Ile Leu Leu Gly         #     60                                                                      - Val Ile Gly Asn Ser Leu Val Ile His Val Va - #l Ile Lys Phe Lys Ser         # 80                                                                          - Met Arg Thr Val Thr Asn Phe Phe Ile Ala As - #n Leu Ala Val Ala Asp         #                 95                                                          - Leu Leu Val Asn Thr Leu Cys Leu Pro Phe Th - #r Leu Thr Tyr Thr Leu         #           110                                                               - Met Gly Glu Trp Lys Met Gly Pro Val Leu Cy - #s His Leu Val Pro Tyr         #       125                                                                   - Ala Gln Gly Leu Ala Val Gln Val Ser Thr Il - #e Thr Leu Thr Val Ile         #   140                                                                       - Ala Leu Asp Arg His Arg Cys Ile Val Tyr Hi - #s Leu Glu Ser Lys Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Lys Arg Ile Ser Phe Leu Ile Ile Gly Le - #u Ala Trp Gly Ile Ser         #               175                                                           - Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Ar - #g Glu Tyr Ser Leu Ile         #           190                                                               - Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cy - #s Thr Glu Lys Trp Pro         #       205                                                                   - Gly Glu Glu Lys Ser Ile Tyr Gly Thr Val Ty - #r Ser Leu Ser Ser Leu         #   220                                                                       - Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile Il - #e Ser Phe Ser Tyr Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Ile Trp Ser Lys Leu Lys Asn His Val Se - #r Pro Gly Ala Ala Asn         #               255                                                           - Asp His Tyr His Gln Arg Arg Gln Lys Thr Th - #r Lys Met Leu Val Cys         #           270                                                               - Val Val Val Val Phe Ala Val Ser Trp Leu Pr - #o Leu His Ala Phe Gln         #       285                                                                   - Leu Ala Val Asp Ile Asp Ser Gln Val Leu As - #p Leu Lys Glu Tyr Lys         #   300                                                                       - Leu Ile Phe Thr Val Phe His Ile Ile Ala Me - #t Cys Ser Thr Phe Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser As - #n Tyr Arg Lys Ala Phe         #               335                                                           - Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu As - #p Ala Ile His Ser Glu         #           350                                                               - Val Ser Val Thr Phe Lys Ala Lys Lys Asn Le - #u Glu Val Arg Lys Asn         #       365                                                                   - Ser Gly Pro Asn Asp Ser Phe Thr Glu Ala Th - #r Asn Val                     #   380                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1556 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 211..1353                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GTTGTTAACA GACTCGTGTA AAGGATTTGC TTTATGGAGC TTTTATGAGA TC - #TGTGGTGT         60                                                                          - GATGAATCAG AACACAGCTA CGCAGAGGAG CTCAGCCTAA ACTAAATCAA CC - #CCTTTAGG        120                                                                          - ATGGTTCTCT GTTTCACTAA CTTTTTTTAA TGTCGTTTTC TGTTATAGAT TC - #TTGTGCTA        180                                                                          - TCTGCAGGCC AAATTGGAAC TGAGGTGAAG ATG GGC CCA TTA GG - #T GCA GAG GCA         234                                                                          #Pro Leu Gly Ala Glu Ala                                                      #              5  1                                                           - GAT GAG AAT CAA ACT GTA GAA GTG AAA GTG GA - #A CTC TAT GGG TCG GGG          282                                                                          Asp Glu Asn Gln Thr Val Glu Val Lys Val Gl - #u Leu Tyr Gly Ser Gly           #     20                                                                      - CCC ACC ACT CCT AGA GGT GAG TTG CCC CCT GA - #T CCA GAG CCG GAG CTC          330                                                                          Pro Thr Thr Pro Arg Gly Glu Leu Pro Pro As - #p Pro Glu Pro Glu Leu           # 40                                                                          - ATA GAC AGC ACC AAA CTG GTT GAG GTG CAG GT - #G GTC CTT ATA CTG GCC          378                                                                          Ile Asp Ser Thr Lys Leu Val Glu Val Gln Va - #l Val Leu Ile Leu Ala           #                 55                                                          - TAT TGT TCC ATC ATC TTG CTG GGC GTA GTT GG - #C AAC TCT CTG GTA ATC          426                                                                          Tyr Cys Ser Ile Ile Leu Leu Gly Val Val Gl - #y Asn Ser Leu Val Ile           #             70                                                              - CAT GTG GTG ATC AAA TTC AAG AGC ATG CGC AC - #A GTA ACC AAC TTT TTT          474                                                                          His Val Val Ile Lys Phe Lys Ser Met Arg Th - #r Val Thr Asn Phe Phe           #         85                                                                  - ATT GCC AAC CTG GCT GTG GCG GAT CTT TTG GT - #G AAC ACC CTG TGC CTG          522                                                                          Ile Ala Asn Leu Ala Val Ala Asp Leu Leu Va - #l Asn Thr Leu Cys Leu           #    100                                                                      - CCA TTC ACT CTT ACC TAT ACC TTG ATG GGG GA - #G TGG AAA ATG GGT CCA          570                                                                          Pro Phe Thr Leu Thr Tyr Thr Leu Met Gly Gl - #u Trp Lys Met Gly Pro           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - GTT TTG TGC CAT TTG GTG CCC TAT GCC CAG GG - #T CTG GCA GTA CAA GTG          618                                                                          Val Leu Cys His Leu Val Pro Tyr Ala Gln Gl - #y Leu Ala Val Gln Val           #               135                                                           - TCC ACA ATA ACT TTG ACA GTC ATT GCT TTG GA - #C CGA CAT CGT TGC ATT          666                                                                          Ser Thr Ile Thr Leu Thr Val Ile Ala Leu As - #p Arg His Arg Cys Ile           #           150                                                               - GTC TAC CAC CTG GAG AGC AAG ATC TCC AAG CA - #A ATC AGC TTC CTG ATT          714                                                                          Val Tyr His Leu Glu Ser Lys Ile Ser Lys Gl - #n Ile Ser Phe Leu Ile           #       165                                                                   - ATT GGC CTG GCG TGG GGT GTC AGC GCT CTG CT - #G GCA AGT CCC CTT GCC          762                                                                          Ile Gly Leu Ala Trp Gly Val Ser Ala Leu Le - #u Ala Ser Pro Leu Ala           #   180                                                                       - ATC TTC CGG GAG TAC TCA CTG ATT GAG ATT AT - #T CCT GAC TTT GAG ATT          810                                                                          Ile Phe Arg Glu Tyr Ser Leu Ile Glu Ile Il - #e Pro Asp Phe Glu Ile           185                 1 - #90                 1 - #95                 2 -       #00                                                                           - GTA GCC TGT ACT GAG AAA TGG CCC GGG GAG GA - #G AAG AGT GTG TAC GGT          858                                                                          Val Ala Cys Thr Glu Lys Trp Pro Gly Glu Gl - #u Lys Ser Val Tyr Gly           #               215                                                           - ACA GTC TAC AGC CTT TCC ACC CTG CTA ATC CT - #C TAC GTT TTG CCT CTG          906                                                                          Thr Val Tyr Ser Leu Ser Thr Leu Leu Ile Le - #u Tyr Val Leu Pro Leu           #           230                                                               - GGC ATC ATA TCT TTC TCC TAC ACC CGG ATC TG - #G AGT AAG CTA AAG AAC          954                                                                          Gly Ile Ile Ser Phe Ser Tyr Thr Arg Ile Tr - #p Ser Lys Leu Lys Asn           #       245                                                                   - CAC GTT AGT CCT GGA GCT GCA AGT GAC CAT TA - #C CAT CAG CGA AGG CAC         1002                                                                          His Val Ser Pro Gly Ala Ala Ser Asp His Ty - #r His Gln Arg Arg His           #   260                                                                       - AAA ACG ACC AAA ATG CTC GTG TGC GTG GTA GT - #G GTG TTT GCA GTC AGC         1050                                                                          Lys Thr Thr Lys Met Leu Val Cys Val Val Va - #l Val Phe Ala Val Ser           265                 2 - #70                 2 - #75                 2 -       #80                                                                           - TGG CTG CCC CTC CAT GCC TTC CAA CTT GCT GT - #G GAC ATC GAC AGC CAT         1098                                                                          Trp Leu Pro Leu His Ala Phe Gln Leu Ala Va - #l Asp Ile Asp Ser His           #               295                                                           - GTC CTG GAC CTG AAG GAG TAC AAA CTC ATC TT - #C ACC GTG TTC CAC ATT         1146                                                                          Val Leu Asp Leu Lys Glu Tyr Lys Leu Ile Ph - #e Thr Val Phe His Ile           #           310                                                               - ATT GCG ATG TGC TCC ACC TTC GCC AAC CCC CT - #T CTC TAT GGC TGG ATG         1194                                                                          Ile Ala Met Cys Ser Thr Phe Ala Asn Pro Le - #u Leu Tyr Gly Trp Met           #       325                                                                   - AAC AGC AAC TAC AGA AAA GCT TTC CTC TCA GC - #C TTC CGC TGT GAG CAG         1242                                                                          Asn Ser Asn Tyr Arg Lys Ala Phe Leu Ser Al - #a Phe Arg Cys Glu Gln           #   340                                                                       - AGG TTG GAT GCC ATT CAC TCG GAG GTG TCC AT - #G ACC TTC AAG GCT AAA         1290                                                                          Arg Leu Asp Ala Ile His Ser Glu Val Ser Me - #t Thr Phe Lys Ala Lys           345                 3 - #50                 3 - #55                 3 -       #60                                                                           - AAG AAC CTG GAA GTC AAA AAG AAC AAT GGC CT - #C ACT GAC TCT TTT TCA         1338                                                                          Lys Asn Leu Glu Val Lys Lys Asn Asn Gly Le - #u Thr Asp Ser Phe Ser           #               375                                                           - GAG GCC ACC AAC GTG TAAGAATGCT GTGAAAGTAC GTGGGTAAA - #T TGCGACCAGA         1393                                                                          Glu Ala Thr Asn Val                                                                       380                                                               - GTTGCCAACC TGGTTAGGGA AGGTTTTCTG GCTAGTGCAT GCCACCTCCC AT - #TGTATTGA       1453                                                                          - CCCTAAAAGC ATCAGAGTGG AAGCCCCAGC GGTATTGTTC CTGGAAAACT GG - #CTGGAAGA       1513                                                                          #                 155 - #6GCTGTGGC GCAACGTTCT GAT                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 381 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Gly Pro Leu Gly Ala Glu Ala Asp Glu As - #n Gln Thr Val Glu Val         #                 15                                                          - Lys Val Glu Leu Tyr Gly Ser Gly Pro Thr Th - #r Pro Arg Gly Glu Leu         #             30                                                              - Pro Pro Asp Pro Glu Pro Glu Leu Ile Asp Se - #r Thr Lys Leu Val Glu         #         45                                                                  - Val Gln Val Val Leu Ile Leu Ala Tyr Cys Se - #r Ile Ile Leu Leu Gly         #     60                                                                      - Val Val Gly Asn Ser Leu Val Ile His Val Va - #l Ile Lys Phe Lys Ser         # 80                                                                          - Met Arg Thr Val Thr Asn Phe Phe Ile Ala As - #n Leu Ala Val Ala Asp         #                 95                                                          - Leu Leu Val Asn Thr Leu Cys Leu Pro Phe Th - #r Leu Thr Tyr Thr Leu         #           110                                                               - Met Gly Glu Trp Lys Met Gly Pro Val Leu Cy - #s His Leu Val Pro Tyr         #       125                                                                   - Ala Gln Gly Leu Ala Val Gln Val Ser Thr Il - #e Thr Leu Thr Val Ile         #   140                                                                       - Ala Leu Asp Arg His Arg Cys Ile Val Tyr Hi - #s Leu Glu Ser Lys Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Lys Gln Ile Ser Phe Leu Ile Ile Gly Le - #u Ala Trp Gly Val Ser         #               175                                                           - Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Ar - #g Glu Tyr Ser Leu Ile         #           190                                                               - Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cy - #s Thr Glu Lys Trp Pro         #       205                                                                   - Gly Glu Glu Lys Ser Val Tyr Gly Thr Val Ty - #r Ser Leu Ser Thr Leu         #   220                                                                       - Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile Il - #e Ser Phe Ser Tyr Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Ile Trp Ser Lys Leu Lys Asn His Val Se - #r Pro Gly Ala Ala Ser         #               255                                                           - Asp His Tyr His Gln Arg Arg His Lys Thr Th - #r Lys Met Leu Val Cys         #           270                                                               - Val Val Val Val Phe Ala Val Ser Trp Leu Pr - #o Leu His Ala Phe Gln         #       285                                                                   - Leu Ala Val Asp Ile Asp Ser His Val Leu As - #p Leu Lys Glu Tyr Lys         #   300                                                                       - Leu Ile Phe Thr Val Phe His Ile Ile Ala Me - #t Cys Ser Thr Phe Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser As - #n Tyr Arg Lys Ala Phe         #               335                                                           - Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu As - #p Ala Ile His Ser Glu         #           350                                                               - Val Ser Met Thr Phe Lys Ala Lys Lys Asn Le - #u Glu Val Lys Lys Asn         #       365                                                                   - Asn Gly Leu Thr Asp Ser Phe Ser Glu Ala Th - #r Asn Val                     #   380                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1200 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 55..1200                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - TTTCTGTTAT AGATTCTTGT GCTATCTGCA GGCCAAATTG GAACTGAGGT GA - #AG ATG           57                                                                          #      Met                                                                    #        1                                                                    - GGC CCA TTA GGT GCA GAG GCA GAT GAG AAT CA - #A ACT GTA GAA GTG AAA          105                                                                          Gly Pro Leu Gly Ala Glu Ala Asp Glu Asn Gl - #n Thr Val Glu Val Lys           #              15                                                             - GTG GAA TTC TAT GGG TCG GGG CCC ACC ACT CC - #T AGA GGT GAG TTG CCC          153                                                                          Val Glu Phe Tyr Gly Ser Gly Pro Thr Thr Pr - #o Arg Gly Glu Leu Pro           #         30                                                                  - CCT GAT CCA GAG CCG GAG CTC ATA GAC AGC AC - #C AAA CTG GTT GAG GTG          201                                                                          Pro Asp Pro Glu Pro Glu Leu Ile Asp Ser Th - #r Lys Leu Val Glu Val           #     45                                                                      - CAG GTG GTC CTT ATA CTG GCC TAT TGT TCC AT - #C ATC TTG CTG GGC GTA          249                                                                          Gln Val Val Leu Ile Leu Ala Tyr Cys Ser Il - #e Ile Leu Leu Gly Val           # 65                                                                          - GTT GGC AAC TCT CTG GTA ATC CAT GTG GTG AT - #C AAA TTC AAG AGC ATG          297                                                                          Val Gly Asn Ser Leu Val Ile His Val Val Il - #e Lys Phe Lys Ser Met           #                 80                                                          - CGC ACA GTA ACC AAC TTT TTT ATT GCC AAC CT - #G GCT GTG GCG GAT CTT          345                                                                          Arg Thr Val Thr Asn Phe Phe Ile Ala Asn Le - #u Ala Val Ala Asp Leu           #             95                                                              - TTG GTG AAC ACC CTG TGC CTG CCA TTC ACT CT - #T ACC TAT ACC TTG ATG          393                                                                          Leu Val Asn Thr Leu Cys Leu Pro Phe Thr Le - #u Thr Tyr Thr Leu Met           #       110                                                                   - GGG GAG TGG AAA ATG GGT CCA GTT TTG TGC CA - #T TTG GTG CCC TAT GCC          441                                                                          Gly Glu Trp Lys Met Gly Pro Val Leu Cys Hi - #s Leu Val Pro Tyr Ala           #   125                                                                       - CAG GGT CTG GCA GTA CAA GTG TCC ACA ATA AC - #T TTG ACA GTC ATT GCT          489                                                                          Gln Gly Leu Ala Val Gln Val Ser Thr Ile Th - #r Leu Thr Val Ile Ala           130                 1 - #35                 1 - #40                 1 -       #45                                                                           - TTG GAC CGA CAT CGT TGC ATT GTC TAC CAC CT - #G GAG AGC AAG ATC TCC          537                                                                          Leu Asp Arg His Arg Cys Ile Val Tyr His Le - #u Glu Ser Lys Ile Ser           #               160                                                           - AAG CAA ATC AGC TTC CTG ATT ATT GGC CTG GC - #G TGG GGT GTC AGC GCT          585                                                                          Lys Gln Ile Ser Phe Leu Ile Ile Gly Leu Al - #a Trp Gly Val Ser Ala           #           175                                                               - CTG CTG GCA AGT CCC CTT GCC ATC TTC CGG GA - #G TAC TCA CTG ATT GAG          633                                                                          Leu Leu Ala Ser Pro Leu Ala Ile Phe Arg Gl - #u Tyr Ser Leu Ile Glu           #       190                                                                   - ATT ATT CCT GAC TTT GAG ATT GTA GCC TGT AC - #T GAG AAA TGG CCC GGG          681                                                                          Ile Ile Pro Asp Phe Glu Ile Val Ala Cys Th - #r Glu Lys Trp Pro Gly           #   205                                                                       - GAG GAG AAG AGT GTG TAC GGT ACA GTC TAC AG - #C CTT TCC ACC CTG CTA          729                                                                          Glu Glu Lys Ser Val Tyr Gly Thr Val Tyr Se - #r Leu Ser Thr Leu Leu           210                 2 - #15                 2 - #20                 2 -       #25                                                                           - ATC CTC TAC GTT TTG CCT CTG GGC ATC ATA TC - #T TTC TCC TAC ACC CGG          777                                                                          Ile Leu Tyr Val Leu Pro Leu Gly Ile Ile Se - #r Phe Ser Tyr Thr Arg           #               240                                                           - ATC TGG AGT AAG CTA AAG AAC CAC GTT AGT CC - #T GGA GCT GCA AGT GAC          825                                                                          Ile Trp Ser Lys Leu Lys Asn His Val Ser Pr - #o Gly Ala Ala Ser Asp           #           255                                                               - CAT TAC CAT CAG CGA AGG CAC AAA ATG ACC AA - #A ATG CTC GTG TGC GTG          873                                                                          His Tyr His Gln Arg Arg His Lys Met Thr Ly - #s Met Leu Val Cys Val           #       270                                                                   - GTA GTG GTG TTT GCA GTC AGC TGG CTG CCC CT - #C CAT GCC TTC CAA CTT          921                                                                          Val Val Val Phe Ala Val Ser Trp Leu Pro Le - #u His Ala Phe Gln Leu           #   285                                                                       - GCT GTG GAC ATC GAC AGC CAT GTC CTG GAC CT - #G AAG GAG TAC AAA CTC          969                                                                          Ala Val Asp Ile Asp Ser His Val Leu Asp Le - #u Lys Glu Tyr Lys Leu           290                 2 - #95                 3 - #00                 3 -       #05                                                                           - ATC TTC ACC GTG TTC CAC ATT ATT GCG ATG TG - #C TCC ACC TTC GCC AAC         1017                                                                          Ile Phe Thr Val Phe His Ile Ile Ala Met Cy - #s Ser Thr Phe Ala Asn           #               320                                                           - CCC CTT CTC TAT GGC TGG ATG AAC AGC AAC TA - #C AGA AAA GCT TTC CTC         1065                                                                          Pro Leu Leu Tyr Gly Trp Met Asn Ser Asn Ty - #r Arg Lys Ala Phe Leu           #           335                                                               - TCA GCC TTC CGC TGT GAG CAG AGG TTG GAT GC - #C ATT CAC TCG GAG GTG         1113                                                                          Ser Ala Phe Arg Cys Glu Gln Arg Leu Asp Al - #a Ile His Ser Glu Val           #       350                                                                   - TCC ATG ACC TTC AAG GCT AAA AAG AAC CTG GA - #A GTC AAA AAG AAC AAT         1161                                                                          Ser Met Thr Phe Lys Ala Lys Lys Asn Leu Gl - #u Val Lys Lys Asn Asn           #   365                                                                       #   1200C ACT GAC TCT TTT TCA GAG GCC ACC AA - #C GTG TAA                     Gly Leu Thr Asp Ser Phe Ser Glu Ala Thr As - #n Val  *                        370                 3 - #75                 3 - #80                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  381 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Gly Pro Leu Gly Ala Glu Ala Asp Glu As - #n Gln Thr Val Glu Val         #                 15                                                          - Lys Val Glu Phe Tyr Gly Ser Gly Pro Thr Th - #r Pro Arg Gly Glu Leu         #             30                                                              - Pro Pro Asp Pro Glu Pro Glu Leu Ile Asp Se - #r Thr Lys Leu Val Glu         #         45                                                                  - Val Gln Val Val Leu Ile Leu Ala Tyr Cys Se - #r Ile Ile Leu Leu Gly         #     60                                                                      - Val Val Gly Asn Ser Leu Val Ile His Val Va - #l Ile Lys Phe Lys Ser         # 80                                                                          - Met Arg Thr Val Thr Asn Phe Phe Ile Ala As - #n Leu Ala Val Ala Asp         #                 95                                                          - Leu Leu Val Asn Thr Leu Cys Leu Pro Phe Th - #r Leu Thr Tyr Thr Leu         #           110                                                               - Met Gly Glu Trp Lys Met Gly Pro Val Leu Cy - #s His Leu Val Pro Tyr         #       125                                                                   - Ala Gln Gly Leu Ala Val Gln Val Ser Thr Il - #e Thr Leu Thr Val Ile         #   140                                                                       - Ala Leu Asp Arg His Arg Cys Ile Val Tyr Hi - #s Leu Glu Ser Lys Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Lys Gln Ile Ser Phe Leu Ile Ile Gly Le - #u Ala Trp Gly Val Ser         #               175                                                           - Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Ar - #g Glu Tyr Ser Leu Ile         #           190                                                               - Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cy - #s Thr Glu Lys Trp Pro         #       205                                                                   - Gly Glu Glu Lys Ser Val Tyr Gly Thr Val Ty - #r Ser Leu Ser Thr Leu         #   220                                                                       - Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile Il - #e Ser Phe Ser Tyr Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Ile Trp Ser Lys Leu Lys Asn His Val Se - #r Pro Gly Ala Ala Ser         #               255                                                           - Asp His Tyr His Gln Arg Arg His Lys Met Th - #r Lys Met Leu Val Cys         #           270                                                               - Val Val Val Val Phe Ala Val Ser Trp Leu Pr - #o Leu His Ala Phe Gln         #       285                                                                   - Leu Ala Val Asp Ile Asp Ser His Val Leu As - #p Leu Lys Glu Tyr Lys         #   300                                                                       - Leu Ile Phe Thr Val Phe His Ile Ile Ala Me - #t Cys Ser Thr Phe Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser As - #n Tyr Arg Lys Ala Phe         #               335                                                           - Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu As - #p Ala Ile His Ser Glu         #           350                                                               - Val Ser Met Thr Phe Lys Ala Lys Lys Asn Le - #u Glu Val Lys Lys Asn         #       365                                                                   - Asn Gly Leu Thr Asp Ser Phe Ser Glu Ala Th - #r Asn Val                     #   380                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #              50ATTGGC CTACTGCTCC ATCATCTTGC TTGGGGTAAT                      - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #              50CACCAA GGAGTTGCCA ATTACCCCAA GCAAGATGAT                      - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #45                TGGC TGTGGCAGAT CTTTTGGTGA ACACT                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #45                TAGA CACAGAGTGT TCACCAAAAG ATCTG                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #45                GCCC AGGGCCTGGC AGTACAAGTA TCCAC                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #45                AAGG TGATTGTGGA TACTTGTACT GCCAG                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #45                ATTG GCTTGGCCTG GGGCATCAGT GCCCT                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #45                CTTG CCAGCAGGGC ACTGATGCCC CAGGC                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #45                CTTC CTTGTTGATC TTGTATGTTT TGCCT                           - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #45                AATG CCCAGAGGCA AAACATACAA GATCA                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #45                TGGT GTTTGCGGTC AGCTGGCTGC CTCTC                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #45                GAAG GCATGGAGAG GCAGCCAGCT GACCG                           - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #                47TCCA CATCATCGCC ATGTGCTCCA CTTTTGC                         - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #                47GAAG GGGATTGGCA AAAGTGGAGC ACATGGC                         - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #               25 TGAG ATCAT                                                 - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #                23ACAC CTC                                                   - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #45                TTTG CAGCTCCAGG ACTGACATGG TTCTT                           - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #45                AGGC AGATGAGAAT CAAACTGTAG AAGTG                           - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #45                TTCT CATCTGCCTC TGCACCTAAT GGGCC                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #45                CTTC AAGGCTAAAA AGAACCTGGA AGTCA                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #45                TTTA GCCTTGAAGG TCATGGACAC CTCCG                           __________________________________________________________________________

What is claimed is:
 1. A process for identifying a chemical compoundwhich specifically binds to a human or rat Y2 receptor, which comprisescontacting nonneuronal cells transfected with a vector comprising DNAencoding, and expressing on their cell surface, (i) a human Y2 receptor,wherein the human Y2 receptor has the amino acid sequence shown in FIG.2 (SEQ ID NO: 2) or (ii) a rat Y2 receptor, wherein the rat Y2 receptorhas the amino acid sequence shown in FIG. 8 (SEQ ID NO: 4), with thechemical compound under conditions suitable for binding, and detectingspecific binding of the chemical compound to the human or rat Y2receptor.
 2. A process for identifying a chemical compound whichspecifically binds to a human or rat Y2 receptor, which comprisescontacting a membrane fraction from a cell extract of nonneuronal cellstransfected with a vector comprising DNA encoding, and expressing ontheir cell surface, (i) a human Y2 receptor, wherein the human Y2receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) or(ii) a rat Y2 receptor, wherein the rat Y2 receptor has the amino acidsequence shown in FIG. 8 (SEQ ID NO: 4), with the chemical compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the human or rat Y2 receptor.
 3. A processinvolving competitive binding for identifying a chemical compound whichspecifically binds to a human or rat Y2 receptor, which comprisesseparately contacting nonneuronal cells transfected with a vectorcomprising DNA encoding, and expressing on their cell surface, (i) ahuman Y2 receptor, wherein the human Y2 receptor has the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) a rat Y2 receptor,wherein the rat Y2 receptor has the amino acid sequence shown in FIG. 8(SEQ ID NO: 4), with both the chemical compound and a second chemicalcompound known to bind to the human or rat Y2 receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the human or rat Y2 receptor, a decrease in binding of the secondchemical compound to the human or rat Y2 receptor in the presence of thechemical compound indicating that the chemical compound binds to thehuman or rat Y2 receptor.
 4. A process involving competitive binding foridentifying a chemical compound which specifically binds to a human orrat Y2 receptor, which comprises separately contacting a membranefraction from a cell extract of nonneuronal cells transfected with avector comprising DNA encoding, and expressing on their cell surface,(i) a human Y2 receptor, wherein the human Y2 receptor has the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) a rat Y2 receptor,wherein the rat Y2 receptor has the amino acid sequence shown in FIG. 8(SEQ ID NO: 4), with both the chemical compound and a second chemicalcompound known to bind to the human or rat Y2 receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the human or rat Y2 receptor, a decrease in binding of the secondchemical compound to the human or rat Y2 receptor in the presence of thechemical compound indicating that the chemical compound binds to thehuman or rat Y2 receptor.
 5. A process for determining whether achemical compound specifically binds to and activates a human or rat Y2receptor, which comprises contacting nonneuronal cells producing asecond messenger response and transfected with a vector comprising DNAencoding, and expressing on their cell surface (i) a human Y2 receptorhaving the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) arat Y2 receptor, wherein the rat Y2 receptor has the amino acid sequenceshown in FIG. 8 (SEQ ID NO: 4), with the chemical compound underconditions suitable for activation of the human or rat Y2 receptor, andmeasuring the second messenger response in the presence and in theabsence of the chemical compound, a change in second messenger responsein the presence of the chemical compound indicating that the chemicalcompound activates the human or rat Y2 receptor.
 6. A process fordetermining whether a chemical compound specifically binds to andactivates a human or rat Y2 receptor, which comprises contacting amembrane fraction from a cell extract of nonneuronal cells producing asecond messenger response and transfected with a vector comprising DNAencoding, and expressing on their cell surface (i) a human Y2 receptorhaving the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) arat Y2 receptor, wherein the rat Y2 receptor has the amino acid sequenceshown in FIG. 8 (SEQ ID NO: 4), with the chemical compound underconditions suitable for activation of the human or rat Y2 receptor, andmeasuring the second messenger response in the presence and in theabsence of the chemical compound, a change in second messenger responsein the presence of the chemical compound indicating that the chemicalcompound activates the human or rat Y2 receptor.
 7. A process fordetermining whether a chemical compound specifically binds to andinhibits activation of a human or rat Y2 receptor, which comprisesseparately contacting nonneuronal cells producing a second messengerresponse and transfected with a vector comprising DNA encoding, andexpressing on their cell surface (i) a human Y2 receptor having theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) a rat Y2receptor, wherein the rat Y2 receptor has the amino acid sequence shownin FIG. 8 (SEQ ID NO: 4), with both the chemical compound and a secondchemical compound known to activate the human or rat Y2 receptor, andwith only the second chemical compound, under conditions suitable foractivation of the human or rat Y2 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in second messenger response in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound indicatingthat the chemical compound inhibits activation of the human or rat Y2receptor.
 8. A process for determining whether a chemical compoundspecifically binds to and inhibits activation of a human or rat Y2receptor, which comprises separately contacting a membrane fraction froma cell extract of nonneuronal cells producing a second messengerresponse and transfected with a vector comprising DNA encoding, andexpressing on their cell surface (i) a human Y2 receptor having theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2) or (ii) a rat Y2receptor, wherein the rat Y2 receptor has the amino acid sequence shownin FIG. 8 (SEQ ID NO: 4), with both the chemical compound and a secondchemical compound known to activate the human or rat Y2 receptor, andwith only the second chemical compound, under conditions suitable foractivation of the human or rat Y2 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in second messenger response in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound indicatingthat the chemical compound inhibits activation of the human or rat Y2receptor.
 9. The process of either of claims 5 or 6, wherein the secondmessenger response comprises adenylate cyclase activity and the changein second messenger response is a decrease in adenylate cyclaseactivity.
 10. The process of either of claims 7 or 8, wherein the secondmessenger response comprises adenylate cyclase activity and the changein second messenger response is a smaller decrease in the level ofadenylate cyclase activity in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound.
 11. The process of claim 5, wherein the secondmessenger response comprises intracellular calcium levels and the changein second messenger response is an increase in intracellular calciumlevels.
 12. The process of claim 7, wherein the second messengerresponse comprises intracellular calcium levels and the change in secondmessenger response is a smaller increase in the level of intracellularcalcium in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.
 13. The process of any one of claims 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12, wherein the nonneuronal cell is a mammalian cell. 14.The process of claim 13, wherein the mammalian cell is a COS-7 cell, anNIH-3T3 cell, a 293 human embryonic kidney cell, or an LM(tk-) cell.